Microbial Pathogenesis 1990 ; 8 : 189-196
Relationship between intracellular survival in macrophages and pathogenicity of Streptococcus suis type 2 isolates A . E . Williams* Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, U.K. (Received October 9, 1989 ; accepted in revised form December 16, 1989)
Williams, A . E . (Dept of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, U .K .) . Relationship between intracellular survival in macrophages and pathogenicity of Streptococcus suis type 2 isolates . Microbial Pathogenesis 1990; 8 : 189-196 . Naturally-occurring Streptococcus suis type 2 meningitis affects pigs and man ; experimental models of the disease have also been established in pigs and mice . A sustained, high-level bacteraemia is an important phase preceding the development of S . suis type 2 meningitis . The main cellular clearance mechanism for circulating bacteria is the resident hepatic and splenic macrophages . The interaction between various isolates of S . suis type 2 and murine macrophages was investigated to determine whether there were differences in the outcome of the interaction that would reflect observed differences in pathogenicity . Phagocytosed non-pathogenic isolates were killed whereas intracellular pathogenic organisms survived and replicated within phagosomes in the absence of anti-S. suis type 2 antibody and complement . The addition of anti-S. suis type 2 antibody and complement to macrophages containing ingested pathogenic organisms resulted in inactivation of the intracellular bacteria . Thus whilst the pathogenicity of S . suis type 2 isolates may be related to an ability to survive within macrophages, immunity to S . suis type 2 meningitis may result from anti-S . suis type 2 antibody preventing pathogenic organisms surviving within macrophages . Key words : Streptococcus suis; pathogenicity ; macrophages .
Introduction Streptococcus suis type 2 is the commonest cause of bacterial meningitis in pigs in 2, and experimental models have been the U .K .' It also causes disease in man established in pigs and mice . Many studies of the interaction in vitro of S . suis type 2 and polymorphonuclear cells (polymorphs) have shown that non-pathogenic isolates are readily phagocytosed in the absence of type-specific antibody and complement whereas both of these are required for efficient phagocytosis of pathogenic (encapsulated) isolates ; phagocytosed organisms are rapidly killed by polymorphs ." in contrast, few investigations have dealt with the interaction of S . suis type 2 with mononuclear phagocytes . The only studies performed to date have failed to demonstrate phagocytosis or killing of capsulated S . suis type 2 by monocytes in mixed peripheral blood mononuclear cell preparations, even in the presence of type-specific antibody .' *Author to whom correspondence should be addressed : A. E . Williams, present address : Department of Paediatrics, Level 4, John Radcliffe Hospital II, Headington, Oxford OX3 9DU, U .K . 0882-4010/90/030189+08 $03 .00/0
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Observations made on ultrastructural examination of meningeal exudate present in pigs with S . suis type 2 meningitis have revealed that both polymorphs and macrophages are capable of phagocytosing pathogenic S . suis type 2 organisms in vivo in pigs lacking anti-S . suis type 2 antibodies .' However, the outcome of the interaction differs ; multiple organisms, many showing cross-wall formation, are found within macrophages but not within polymorphs,' indicating possible intracellular survival of pathogenic S . suis type 2 organisms within macrophages . The ability of an isolate of a meningitis-causing bacteria to cause and maintain bacteraemia is a major determinant in the pathogenesis of meningitis since a highlevel, persistent bacteraemia usually precedes the onset of bacterial meningitis . ? 10 The mononuclear phagocytes in the liver and spleen play a major role in the clearance of .'"' Thus the outcome of the interaction circulating organisms from the bloodstream of circulating organisms with these phagocytes is crucial to the development of a persistent bacteraemia and meningitis . This paper describes a series of studies of the interaction of macrophages with pathogenic and non-pathogenic isolates of S . suis type 2 in vitro . Murine elicited peritoneal macrophages were used, a previous study having demonstrated that many of the features of porcine S, suis type 2 meningitis are reproduced in mice ." An assay to examine phagocytosis and subsequent intracellular fate of ingested S . 2 organisms by macrophages was developed by adapting an acridine orangecrystal violet technique ."- " Vital staining with acridine orange (AO) is a very sensitive method for detecting ingested bacteria 18-22 and also allows differentiation between live and dead intracellular organisms since dead bacteria bind more AO dye and fluoresce red (metachromasy), whilst living bacteria bind less dye and fluoresce green (orthochromasy) .2023 2s A shift in fluorescence towards metachromasia, the 'Strugger effect', may therefore be used as a test for viability of organisms . 19z0 The use of AO to assess phagocytosis and intracellular killing of bacteria by leucocytes is improved by the use of crystal violet (CV) as a second stain because CV quenches the fluorescence of surface-adherent, extracellular bacteria, whilst the fluorescence of intracellular bacteria remains unaffected .", " This quenching does not alter cellular AO staining and is unaffected by repeated washing .' 1,2' Thus the use of CV prevents the erroneous
suis type
counting of extracellular bacteria as being intracellular . The AO-CV technique, therefore, offers a simple but very sensitive method that allows the simultaneous, quantitative analysis of bacterial uptake and intracellular killing by phagocytic cells in vitro, in which both stages-phagocytosis and subsequent intracellular killing-can be distinguished .
Results The protocol used to obtain peritoneal cell suspensions reliably gave suspensions of approximately 2 .5±0 .14X10 7 cells/ml (>95% macrophages) . The mean number of adherent macrophages on each coverslip was 1 .93±0 .12X10' . An organism to macrophage ratio of 10 :1 was chosen because significant results were obtained and were easy to observe . In individual experiments, the ratio ranged from 7 .3 :1 to 16 .41 ; however, consistent results were obtained over this range . The percentage of cells containing ingested organisms after the first 60 min of incubation was similar for all isolates and was 8 .67% (range : 6-13% ; SD = 1 .78) . Those cells containing phagocytosed bacteria contained an average of 2 .6-6 .8 organisms at this time (Table 1 ) . On reincubation, the number of cells containing bacteria did not change significantly (data not shown) . However, the mean number of phagocytosed organisms increased in the case of pathogenic isolates and remained
Survival of pathogenic S . suis type 2 in macrophages
191
Table 1 Mean (and SE) number of ingested S . suis type 2 organisms within murine elicited peritoneal macrophagesreincubation in RPMI 1640 with 10% heat-inactivated FCS Minutes of reincubation Isolate Pathogenic' P1/7 R75/L, H11 /1 NEW GB1 Non-pathogenic' TD/10 H11/1OLD
0
5 .2 4 .2 2 .8 5.7
(0 .5) (0 .7) (0 .1) (0 .4)
4.0 (0 .6) 3.0 (0 .2)
30
8.9 7 .2 4.5 9 .1
(0 .9) (0 .5) (0 .5) (1 .0)
2 .9 (0 .3) 3 .3 (0 .2)
60
11 .3 10 .7 6 .5 10 .3
(1 .2) (0 .2) (0 .9) (0 .3)
3 .1 (0 .4) 3 .7 (0 .3)
90
12 .9 11 .5 9 .0 12 .0
(1 .5) (0 .4) (1 .0) (0 .5)
3 .3 (0 .5) 3 .6 (0 .5)
Only cells with ingested organisms are represented in these results . 'See Table 3 .
constant for non-pathogenic isolates (Table 1 ) . There were no statistically significant differences between the results for the two non-pathogenic isolates, nor were there significant differences between the results for the pathogenic isolates . The differences between the mean number of intracellular pathogenic and non-pathogenic isolates (in cells with ingested bacteria) after 90 min of reincubation were statistically significant (P < 0 .001, unpaired Student's t-test) . AO staining revealed a further difference between pathogenic and non-pathogenic isolates . After the initial 60 min of incubation during which phagocytosis occurred, the ratio of green or pale orange (live) to red (dead) organisms was consistently 1520 :1 for pathogenic isolates and 4-7 :1 for non-pathogenic isolates . After the further 90 min of reincubation this ratio was still the same for pathogenic isolates but was 0 .05-0 .08 :1 for non-pathogenic isolates . There was a difference in the morphology of macrophages associated with the phagocytosis of pathogenic organisms . Initially the morphology of the macrophages that interacted with pathogenic or non-pathogenic isolates was similar ; cells showed green nuclei, orange cytoplasm and occasional deep orange-red cytoplasmic granules larger than bacteria in size . Macrophages interacting with non-pathogenic isolates retained this appearance at the end of the test, although cytoplasmic granules were less evident . This appearance was also retained by macrophages incubated with pathogenic isolates that had not phagocytosed organisms . However some, but not all, macrophages containing ingested organisms of pathogenic isolates showed paler cytoplasmic staining and large vacuoles after 60 and 90 min reincubation .
Experiments with rabbit sera The results of experiments in which macrophages containing intracellular pathogenic organisms were reincubated in a medium (R PMI 1640) containing either normal rabbit serum or complement-depleted hyperimmune rabbit serum during the reincubation period were indistinguishable from those obtained in experiments using foetal calf serum (see above) and in assays performed without serum (data not shown), i .e . the mean number of intracellular organisms in cells with ingested bacteria increased throughout this period (Table 2) and retained the orthochromasy of live organisms . In contrast, the results of experiments in which macrophages containing intracellular pathogenic organisms were reincubated in RPMI 1640 containing hyperimmune serum were similar to those obtained for the non-pathogenic isolates-the mean number of
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Table 2 Mean (and SE) number of ingested pathogenic S. suis type 2 organisms (P1 /7) within murine elicited peritoneal macrophages reincubation with rabbit sera Minutes of reincubation Type of serum Normal rabbit serum Complement-depleted hyperimmune rabbit serum Hyperimmune rabbit serum
0
30
60
3 .7 (0 .3) 3 .5 (0 .1) 3 .4 (0 .3)
6 .3 (0.2) 6 .2 (0.2) 3 .3 (0.4)
8 .8 (0.3) 9 .1 (0.2) 3 .6 (0.6)
90 11 .4 (0 .5) 11 .8 (0 .5) 3 .4 (0 .4)
Only cells with ingested organisms are represented in these results . For concentration of sera in RPMI, see text .
intracellular organisms did not increase (Table 2) and the predominant fluorescence characteristics changed to the metachromasy of dead bacteria . Discussion and conclusions In these experiments, the AO-CV technique developed by previous workers' 5,17,19,20 was modified to examine the phagocytic index and subsequent bactericidal activity of macrophages for various isolates of S . suis type 2 . The modification of removing noningested organisms after an initial period of incubation and then reincubating the cell preparations allowed the sequential phases of phagocytosis and intracellular killing to be studied separately . Preliminary studies omitting CV staining had shown that not all extracellular organisms were removed by washing after the initial 60 min incubation period ; rare extracellular bacteria (< 0 .2% total bacteria observed) were seen . However, the proportion of cells containing intracellular organisms remained constant throughout the reincubation period and the mean number of ingested non-pathogenic organisms per cell did not increase (see Table 1 ) . Thus further phagocytosis of extracellular organisms during the 90 min reincubation period did not occur to a significant degree and therefore one can conclude that any observed increase in the number of intracellular organisms during the reincubation period was due to bacterial replication . These studies showed a difference in the nature of the interaction of elicited macrophages with pathogenic and non-pathogenic isolates of S . suis type 2 in vitro ; pathogenic isolates replicated within these cells whereas ingested non-pathogenic organisms were killed . Furthermore, some macrophages containing replicating organisms showed morphological changes consistent with cellular degeneration after 6090 min of reincubation, indicating that such replication may eventually cause the destruction of the phagocyte . These findings therefore substantiate a conclusion previously drawn from an ultrastructural examination of meningeal exudate in cases of S. suis type 2 meningitis that bacteria may replicate within macrophages .' The 100% correlation between intracellular replication within macrophages reported here and pathogenicity in vivo is of particular significance because it indicates that some bacterial factor (or factors) determines the outcome of macrophage-S . suis type 2 interactions that reflect pathogenicity . It is well recognized that the level and duration of bacteraemia are major determinants in the pathogenesis of meningitis . 7-1° The most important cells responsible for clearance of circulating organisms in the non-immune animal are the macrophages of the reticulo-endothelial system (RES) in the liver and spleen .' 1-13 Thus the functional status of the RES is important in reducing the number of circulating organisms available to infect various sites (e .g . the CSF) and therefore in reducing the incidence of meningitis .' 1-13 The results of the present studies suggest that circulating pathogenic
Survival of pathogenic S . suis type 2 in macrophages
193
S. suis type 2 organisms are unlikely to be cleared by the RES in pigs lacking antibodies against S . suis type 2, and so a persistent bacteraemia will develop, favouring the development of meningitis . In contrast, circulating non-pathogenic organisms would be eliminated . The importance of the outcome of the interaction of circulating S . suis type 2 organisms with mononuclear phagocytes is dramatically demonstrated by the observation that organisms of encapsulated 'non-pathogenic' isolates-that are rapidly cleared from the circulation of mice and of pigs lacking antibodies to S . suis type 2 5 ' 14-cause meningitis when inoculated into the cisterna magnum but do not cause naturally-occurring meningitis nor meningitis when inoculated intravenously .' .'
The role of antibody in S . suis type 2-macrophage interactions The effect of specific antibody in macrophage-S. suis type 2 interactions was also investigated in the present studies . Only the role of type-specific antibody in intracellular killing of pathogenic S . suis type 2 isolates was examined because problems interpreting results obtained in preliminary experiments due to clumping of organisms in hyperimmune (rabbit) and immune (pig) sera prevented accurate determination of the role of such antibody in phagocytosis . The present studies showed that, in the presence of complement factors, anti-S . suis type 2 antibody mediated intracellular killing of phagocytosed pathogenic organisms by macrophages . Intracellular organisms were killed only in the presence of both antibody and complement factors, a result consistent with previous investigations of the interaction of S . suis type 2 with polymorphs ."' The present results therefore suggest that phagocytosed pathogenic organisms would be killed by the macrophages of the RES in pigs with anti-S . suis type 2 antibodies . Since the level and duration of bacteraemia are important factors in the pathogenesis of meningitis (see above), the presence of type-specific antibody would promote the clearance of a S . suis type 2 bacteraemia and so meningitis would be less likely to develop . It is pertinent to note here that in the present studies anti-S . suis type 2 antibody was added to the macrophage preparations after phagocytosis had occurred (after the first phase of the assay) and therefore killing of ingested P1 /7 (pathogenic) organisms was mediated by a non-opsonic mechanism of the antibody . A similar phenomenon of intracellular bactericidal activity of an antibody entering the phagocyte after ingestion of organisms had occurred has also been recorded for Group B Streptococcus type Ic . 27-30 Whilst it has been demonstrated that such antibody attaches to Fc receptors on the neutrophil surface membrane, enters the cell and penetrates into the phagolysosome to coat the bacterial surface, 29 ' 30 the mechanisms by which such antibody enters the phagolysosome and mediates killing requires further investigation .
Materials and methods
Macrophages . Elicited murine peritoneal macrophages were obtained using standard procedures ." Two, 2-4-month-old ICI mice of either sex were used in each individual assay . The mice were inoculated i .p . with 1 .5 ml of aged sterile broth [3% thioglycollate broth (Brewer) ; Oxoid] and were killed 4 days later . Macrophages were recovered from the freshly-killed mice by washing the peritoneal cavity with 1 .0 ml Ca, Mg-free HBSS, pooled, pelleted by centrifugation at 600xg for 15 min, washed (by centrifugation) three times in Ca, Mg-free HESS and finally resuspended in 2 .0 ml culture medium [RPMI 1640 with glutamine and 20 mm HEPES, lacking bicarbonate and antibiotics, and supplemented with 10% heat-inactivated foetal calf serum ; Flow Laboratories (Med/FCS)] pre-warmed to 37°C ; 0 .2 ml of the macrophage suspension was placed on each of nine 19 mm circular glass coverslips and these were incubated in a humid atmosphere for 45 min at 37°C in 5% CO 2 . Non-adherent cells were removed by two washings with Ca, Mg-free HBSS ; these washings were retained . The remaining 0.2 ml of the cell suspension was used to determine, using a Coulter counter, the number of cells in the
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A . E . Williams Table 3 Isolate P1 /7 R75/L 1 H11/1NEW GB1 TD/10 H11 /1 OLD
Isolates of S. suis type 2 used in these studies Source Pig meningitis Human meningitis H11/1OLDb Pig meningitis Pig tonsil (asymptomatic carrier) Pig tonsil (asymptomatic carrier)
Pathogenicity'
Reference 4 4,32 6 6 5,33 6
± Pathogenicity following i .v . inoculation of 109 cfu organisms in pigs : +++, meningitis, arthritis, serositis; -, none or mild transient lameness . 'Storage of H11/1OLD in liquid blood-broth culture at 4±C for 2-3 weeks results in the development of haemolytic activity, which is maintained thereafter on repeated passage, and pathogenicity .'
suspension placed on the coverslips and to make smears to determine the proportion of macrophages in the cell suspension . To ensure reproducibility of the macrophage preparations, the number of macrophages adherent to a coverslip in an assay was estimated by determining the number of cells in 0 .2 ml of the suspension placed on each coverslip (see above) and measuring the total volume of Ca, Mg-free HBSS used to wash the coverslips and the number of cells (non-adherent cells) in the collected washings .
Bacteria . Four pathogenic isolates and two encapsulated, non-pathogenic isolates of S . suis type 2 were used in these studies ; the virulence of these isolates had been previously established by i .v . inoculation of susceptible pigs` (Table 3) . Organisms in mid-exponential phase of growth were obtained from a 7-9 h culture of S . suis type 2 in Todd-Hewitt Broth, sub-cultured from a 12 h culture in 10% horse-blood broth . Twenty millilitres of growth in Todd-Hewitt Broth was pelleted by centrifugation and resuspended in a similar volume of McCoy's 5A Salt Solution (Flow Laboratories) . An estimation of the concentration of organisms present was made using a Thoma bacterial cell counting chamber and the suspension of organisms in McCoy's 5A adjusted so that 0 .2 ml contained 10 times more live organisms the number of macrophages adherent on the coverslips in that assay [about 90% of the bacteria in a 7-9 h culture of S. suis type 2 organisms in Todd-Hewitt Broth are viable (M . R . Enright, personal communication ; A . Williams, personal observations)] . An accurate determination of the concentration of live bacteria in the final suspension was made using a pour-plate method .' Stains . Acridine orange (Sigma) was used at a concentration of 14 .4 mg/100 ml sterile Gey's Balanced Salt solution and crystal violet (Hopkin and Williams) at a concentration of 50 mg/100 ml of 0 .15M (0 .85%) sterile saline . These stains, and all other salt solutions used, were adjusted to a pH of 7 .2 using 7 .5% sodium bicarbonate and warmed to 37±C before use . Assay . Two isolates were tested in each individual assay and each isolate was tested at least three times . In each assay, four coverslips were incubated with 0 .2 ml of bacterial suspension of one isolate and four more with the other . In most cases, one pathogenic and one nonpathogenic isolate were used in each assay to ensure that the behaviour of the macrophages did not vary between assays . A ninth coverslip acted as a control for macrophage viability throughout the assay and was incubated with 0 .2 ml sterile McCoy's 5A solution . The coverslips were incubated in a humid atmosphere for 60 min at 37±C in 5% CO 2 . After this time, the coverslips were washed five times with Ca, Mg-free HBSS to remove extracellular organisms, covered with warmed Med/FCS and reincubated for a further 0, 30, 60, or 90 min in similar conditions . At each time interval, one coverslip from each of the two isolates being tested was washed three times with Ca, Mg-free HBSS, stained with acridine orange for 45 s, washed twice, stained with crystal violet, washed twice, placed cell-side downwards onto a clean glass microscope slide and sealed with nail varnish . The control macrophage preparation was stained after 90 min reincubation . The preparations were viewed under oil at a magnification of x 1000 using a Nikon Optiphot
Survival of pathogenic S. suis type 2 in macrophages
195
microscope with a mercury lamp source, a 450-490 nm excitation filter, a 510 dichroic mirror and a 520 nm barrier filter (Nikon filter cassette B 2 ) . Cells were counted until 50 had been observed to contain intracellular organisms . The proportion of cells containing ingested organisms, the number of organisms in each cell showing phagocytosis and the colour of fluorescence of ingested bacteria were recorded . Studies using rabbit sera . A further series of experiments was performed in which the foetal calf serum in the Med/FCS used during the reincubation period was replaced by fresh rabbit serum . The rabbit serum added was either : normal serum (1 : 5 in RPMI 1640), hyperimmune serum raised against washed, formalin-killed cultures of S . suis type 2 34 (1 :10) or hyperimmune serum previously heated at 56°C for 20 min to remove complement factors (1 :10) . The same pathogenic isolate (P1 /7) was used in all these studies .
The author is pleased to acknowledge the assistance of W . F . Blakemore . This work was supported by funds from the Agricultural and Food Research Council, U .K .
References Clifton-Hadley FA, Alexander TJL . Diagnosis of Streptococcus suis infection in pigs . In Practice (supplement to Vet Rec) 1988 ; 10: 185-7 . 2 . Chau PY, Huang CY, Kay R . Streptococcus suis type 2 meningitis : an important underdiagnosed disease in Hong Kong . Med J Austral 1983 ; 1 : 414-7 . 3 . Arends JP, Zanen HC . Meningitis caused by Streptococcus suis in humans . Rev Infect Dis 1988; 10 : 131-7 . 4 . Clifton- Hadley FA . Studies of Streptococcus suis infection in pigs . PhD thesis, University of Cambridge, 1981 5 . Upton I . The immune response of the pig to infections with Streptococcus suis type 2 . PhD thesis, University of Cambridge, 1986 . 6 . Williams AE . Studies on the pathogenesis of Streptococcus suis type 2 in the pig . PhD thesis, University of Cambridge, 1989 . 7 . Moxon ER, Ostrow PT . Haemophi/us inf/uenzae meningitis in infant rats : role of bacteremia in pathogenesis of age-dependant inflammatory responses in cerebrospinal fluid . J Infect Dis 1977 ; 135 : 303-7 . 8 . Bortolussi R, Ferrieri P, Wannamaker LW . Dynamics of Escherichia coli infection and meningitis in infant rats. Infect Immun 1978 ; 22 : 480-5 . 9 . Ferrieri P, Burke B, Nelson J . Production of bacteremia and meningitis in infant rats with Group B Streptococcal serotypes . Infect Immun 1980 ; 27 : 1023-32 . 10 . Salit IE, Tomalty L . Experimental meningococcal infection in mice : a model for mucosal invasion . Infect Immun 1986 ; 51 : 648-52 . 11 . Weller PF, Smith AL, Smith DH, Anderson P . Role of immunity in the clearance of bacteremia due to Haemophilus influenzae . J Infect Dis 1978 ; 138 : 427-36 . 12 . Moxon ER, Goldthorn JF, Schwartz AD . Haemophilus inf/uenzae infection in rats : effect of splenectomy on bloodstream and meningeal invasion after intravenous and intranasal inoculations . Infect Immun 1980 ; 27 : 872--5 . 13 . Brown EJ, Hosea SW, Frank MM . The role of antibody and complement in the reticuloendothelial clearance of pneumococci from the bloodstream . Rev Infect Dis 1983; 5 [Suppl . 4] : S797-805 . 14 . Williams AE, Blakemore WF, Alexander TJL . A murine model of Streptococcus suis type 2 meningitis in the pig . Res Vet Sci 1988 ; 45 : 394-9 . 15 . Goldner M, Farkas-Himsley H, Kormendy A, Skinner M . Bacterial phagocytosis monitored by fluorescence and extracellular quenching : ingestion and intracellular killing . Lab Med 1983 ; 14 : 291-4 . 16 . Hed J . The extinction of fluorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis . FEMS Microbiol Lett 1977 ; 1 : 357-61 . 17 . Zanetti M, Schmitt M, Lazary S . Bovine leukocyte phagocytosis and bacteria killing monitored by intracellular acridine orange fluorescence and extracellular fluorescence quenching . Vet Immunol Immunopathol 1987 ; 16 : 185-9 . 18 . Feldman WE . Concentration of bacteria in cerebrospinal fluid of patients with bacterial meningitis . J Pediatr 1976 ; 88 : 549-52 . 19 . Smith DL, Rommel F . A rapid micro method for the simultaneous determination of phagocyticmicrobicidal activity of human peripheral blood leukocytes in vitro . J Immunol Meth 1977 ; 17 : 241-7 . 20 . Pantazis CG, Kniker WT . Assessment of blood leukocyte microbial killing by using a new fluorochrome microassay . J Reticuloendothelial Soc 1979 ; 26 : 155-70 . 21 . McCarthy LR, Senne JE . Evaluation of acridine orange stain for detection of microorganisms in blood cultures . J Clin Microbiol 1980 ; 11 : 281-5 .
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22 . Kleinman MB, Reynolds JK, Watts NH, Schreiner RL, Smith JW . Superiority of acridine orange stain versus Gram stain in partially treated bacterial meningitis . J Pediatr 1984 ; 104 : 401-4 . 23 . Strugger S . Fluorescence microscope examination of bacteria in soil . Can J Res 1948; 26(C) : 188-93 . 24 . Ranada SS, Tatake VG, Korgaonkar KS . Effects of ultrasonic radiation in Escherichia co/i B using fluorochrome acridine orange as a vital stain . Nature 1961 ; 189 : 931-2 . 25 . Rigler R . Acridine orange in nucleic acid analysis . Ann N Y Acad Sci 1969 ; 157 : 211-24 . 26 . Pruzanski W, Saito S . Comparative study of phagocytosis and intracellular bactericidal activity of human monocytes and polymorphonuclear cells . Inflammation 1988; 12 : 87-97 . 27 . Cleat PH, Coid CR . Phagocytic and bactericidal activity of human neutrophils against two isolates of Group B Streptococci Type Ic of differing pathogenicity . Br J Exp Path 1981 ; 62 : 393-7 . 28 . Cleat PH, Coid CR . An alternative role for specific antibody in neutrophil bactericidal activity against highly pathogenic Group B Streptococci . Br J Exp Path 1982 ; 63 : 452-7 . 29 . Cleat PH, Coid CR . The mode of action of antibody in neutrophil bactericidal activity against highly pathogenic Group B Streptococci . Med Microbiol Immunol 1984 ; 173 : 65-74 . 30 . Cleat PH, Wells C, Coid CR . Electron microscopic evidence of antibody entry into neutrophils after phagocytosis of highly virulent Group B Streptococci . J Gen Microbiol 1984 ; 130 : 3059-61 . 31 . Stuart AE, Habeshaw JA, Davidson AE . Phagocytes in vitro . In : Weir DM, ed . Handbook of experimental immunology, 3rd edn . Oxford : Blackwell Scientific Publications, 1978; 31 .1-31 .29 . 32 . Elliott SDE, McCarty M, Lancefield RC . Teichoic acids of Group D Streptococci with special reference to strains from pig meningitis . J Exp Med 1977 ; 145: 490-9 . 33 . Clifton-Hadley FA, Alexander TJL, Enright MR, Lindsay HJ . Monitoring herds for Streptococcus suis type 2 : cross-reactions and variance in virulence . Proc 9th Cong Internat Pig Vet Soc, Barcelona 1986 ; 359 . 34 . Clifton-Hadley FA, Alexander TJL, Enright MR . Diagnosis of Streptococcus suis type 2 infection in pigs . Pig Vet Soc Proc 1985 ; 14: 27--34 .
Relationship between intracellular survival in macrophages and pathogenicity of Streptococcus suis type 2 isolates A . E . Williams* Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, U.K. (Received October 9, 1989 ; accepted in revised form December 16, 1989)
Williams, A . E . (Dept of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, U .K .) . Relationship between intracellular survival in macrophages and pathogenicity of Streptococcus suis type 2 isolates . Microbial Pathogenesis 1990; 8 : 189-196 . Naturally-occurring Streptococcus suis type 2 meningitis affects pigs and man ; experimental models of the disease have also been established in pigs and mice . A sustained, high-level bacteraemia is an important phase preceding the development of S . suis type 2 meningitis . The main cellular clearance mechanism for circulating bacteria is the resident hepatic and splenic macrophages . The interaction between various isolates of S . suis type 2 and murine macrophages was investigated to determine whether there were differences in the outcome of the interaction that would reflect observed differences in pathogenicity . Phagocytosed non-pathogenic isolates were killed whereas intracellular pathogenic organisms survived and replicated within phagosomes in the absence of anti-S. suis type 2 antibody and complement . The addition of anti-S. suis type 2 antibody and complement to macrophages containing ingested pathogenic organisms resulted in inactivation of the intracellular bacteria . Thus whilst the pathogenicity of S . suis type 2 isolates may be related to an ability to survive within macrophages, immunity to S . suis type 2 meningitis may result from anti-S . suis type 2 antibody preventing pathogenic organisms surviving within macrophages . Key words : Streptococcus suis; pathogenicity ; macrophages .
Introduction Streptococcus suis type 2 is the commonest cause of bacterial meningitis in pigs in 2, and experimental models have been the U .K .' It also causes disease in man established in pigs and mice . Many studies of the interaction in vitro of S . suis type 2 and polymorphonuclear cells (polymorphs) have shown that non-pathogenic isolates are readily phagocytosed in the absence of type-specific antibody and complement whereas both of these are required for efficient phagocytosis of pathogenic (encapsulated) isolates ; phagocytosed organisms are rapidly killed by polymorphs ." in contrast, few investigations have dealt with the interaction of S . suis type 2 with mononuclear phagocytes . The only studies performed to date have failed to demonstrate phagocytosis or killing of capsulated S . suis type 2 by monocytes in mixed peripheral blood mononuclear cell preparations, even in the presence of type-specific antibody .' *Author to whom correspondence should be addressed : A. E . Williams, present address : Department of Paediatrics, Level 4, John Radcliffe Hospital II, Headington, Oxford OX3 9DU, U .K . 0882-4010/90/030189+08 $03 .00/0
0c
1990 Academic Press Limited
1 90
A . E . Williams
Observations made on ultrastructural examination of meningeal exudate present in pigs with S . suis type 2 meningitis have revealed that both polymorphs and macrophages are capable of phagocytosing pathogenic S . suis type 2 organisms in vivo in pigs lacking anti-S . suis type 2 antibodies .' However, the outcome of the interaction differs ; multiple organisms, many showing cross-wall formation, are found within macrophages but not within polymorphs,' indicating possible intracellular survival of pathogenic S . suis type 2 organisms within macrophages . The ability of an isolate of a meningitis-causing bacteria to cause and maintain bacteraemia is a major determinant in the pathogenesis of meningitis since a highlevel, persistent bacteraemia usually precedes the onset of bacterial meningitis . ? 10 The mononuclear phagocytes in the liver and spleen play a major role in the clearance of .'"' Thus the outcome of the interaction circulating organisms from the bloodstream of circulating organisms with these phagocytes is crucial to the development of a persistent bacteraemia and meningitis . This paper describes a series of studies of the interaction of macrophages with pathogenic and non-pathogenic isolates of S . suis type 2 in vitro . Murine elicited peritoneal macrophages were used, a previous study having demonstrated that many of the features of porcine S, suis type 2 meningitis are reproduced in mice ." An assay to examine phagocytosis and subsequent intracellular fate of ingested S . 2 organisms by macrophages was developed by adapting an acridine orangecrystal violet technique ."- " Vital staining with acridine orange (AO) is a very sensitive method for detecting ingested bacteria 18-22 and also allows differentiation between live and dead intracellular organisms since dead bacteria bind more AO dye and fluoresce red (metachromasy), whilst living bacteria bind less dye and fluoresce green (orthochromasy) .2023 2s A shift in fluorescence towards metachromasia, the 'Strugger effect', may therefore be used as a test for viability of organisms . 19z0 The use of AO to assess phagocytosis and intracellular killing of bacteria by leucocytes is improved by the use of crystal violet (CV) as a second stain because CV quenches the fluorescence of surface-adherent, extracellular bacteria, whilst the fluorescence of intracellular bacteria remains unaffected .", " This quenching does not alter cellular AO staining and is unaffected by repeated washing .' 1,2' Thus the use of CV prevents the erroneous
suis type
counting of extracellular bacteria as being intracellular . The AO-CV technique, therefore, offers a simple but very sensitive method that allows the simultaneous, quantitative analysis of bacterial uptake and intracellular killing by phagocytic cells in vitro, in which both stages-phagocytosis and subsequent intracellular killing-can be distinguished .
Results The protocol used to obtain peritoneal cell suspensions reliably gave suspensions of approximately 2 .5±0 .14X10 7 cells/ml (>95% macrophages) . The mean number of adherent macrophages on each coverslip was 1 .93±0 .12X10' . An organism to macrophage ratio of 10 :1 was chosen because significant results were obtained and were easy to observe . In individual experiments, the ratio ranged from 7 .3 :1 to 16 .41 ; however, consistent results were obtained over this range . The percentage of cells containing ingested organisms after the first 60 min of incubation was similar for all isolates and was 8 .67% (range : 6-13% ; SD = 1 .78) . Those cells containing phagocytosed bacteria contained an average of 2 .6-6 .8 organisms at this time (Table 1 ) . On reincubation, the number of cells containing bacteria did not change significantly (data not shown) . However, the mean number of phagocytosed organisms increased in the case of pathogenic isolates and remained
Survival of pathogenic S . suis type 2 in macrophages
191
Table 1 Mean (and SE) number of ingested S . suis type 2 organisms within murine elicited peritoneal macrophagesreincubation in RPMI 1640 with 10% heat-inactivated FCS Minutes of reincubation Isolate Pathogenic' P1/7 R75/L, H11 /1 NEW GB1 Non-pathogenic' TD/10 H11/1OLD
0
5 .2 4 .2 2 .8 5.7
(0 .5) (0 .7) (0 .1) (0 .4)
4.0 (0 .6) 3.0 (0 .2)
30
8.9 7 .2 4.5 9 .1
(0 .9) (0 .5) (0 .5) (1 .0)
2 .9 (0 .3) 3 .3 (0 .2)
60
11 .3 10 .7 6 .5 10 .3
(1 .2) (0 .2) (0 .9) (0 .3)
3 .1 (0 .4) 3 .7 (0 .3)
90
12 .9 11 .5 9 .0 12 .0
(1 .5) (0 .4) (1 .0) (0 .5)
3 .3 (0 .5) 3 .6 (0 .5)
Only cells with ingested organisms are represented in these results . 'See Table 3 .
constant for non-pathogenic isolates (Table 1 ) . There were no statistically significant differences between the results for the two non-pathogenic isolates, nor were there significant differences between the results for the pathogenic isolates . The differences between the mean number of intracellular pathogenic and non-pathogenic isolates (in cells with ingested bacteria) after 90 min of reincubation were statistically significant (P < 0 .001, unpaired Student's t-test) . AO staining revealed a further difference between pathogenic and non-pathogenic isolates . After the initial 60 min of incubation during which phagocytosis occurred, the ratio of green or pale orange (live) to red (dead) organisms was consistently 1520 :1 for pathogenic isolates and 4-7 :1 for non-pathogenic isolates . After the further 90 min of reincubation this ratio was still the same for pathogenic isolates but was 0 .05-0 .08 :1 for non-pathogenic isolates . There was a difference in the morphology of macrophages associated with the phagocytosis of pathogenic organisms . Initially the morphology of the macrophages that interacted with pathogenic or non-pathogenic isolates was similar ; cells showed green nuclei, orange cytoplasm and occasional deep orange-red cytoplasmic granules larger than bacteria in size . Macrophages interacting with non-pathogenic isolates retained this appearance at the end of the test, although cytoplasmic granules were less evident . This appearance was also retained by macrophages incubated with pathogenic isolates that had not phagocytosed organisms . However some, but not all, macrophages containing ingested organisms of pathogenic isolates showed paler cytoplasmic staining and large vacuoles after 60 and 90 min reincubation .
Experiments with rabbit sera The results of experiments in which macrophages containing intracellular pathogenic organisms were reincubated in a medium (R PMI 1640) containing either normal rabbit serum or complement-depleted hyperimmune rabbit serum during the reincubation period were indistinguishable from those obtained in experiments using foetal calf serum (see above) and in assays performed without serum (data not shown), i .e . the mean number of intracellular organisms in cells with ingested bacteria increased throughout this period (Table 2) and retained the orthochromasy of live organisms . In contrast, the results of experiments in which macrophages containing intracellular pathogenic organisms were reincubated in RPMI 1640 containing hyperimmune serum were similar to those obtained for the non-pathogenic isolates-the mean number of
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Table 2 Mean (and SE) number of ingested pathogenic S. suis type 2 organisms (P1 /7) within murine elicited peritoneal macrophages reincubation with rabbit sera Minutes of reincubation Type of serum Normal rabbit serum Complement-depleted hyperimmune rabbit serum Hyperimmune rabbit serum
0
30
60
3 .7 (0 .3) 3 .5 (0 .1) 3 .4 (0 .3)
6 .3 (0.2) 6 .2 (0.2) 3 .3 (0.4)
8 .8 (0.3) 9 .1 (0.2) 3 .6 (0.6)
90 11 .4 (0 .5) 11 .8 (0 .5) 3 .4 (0 .4)
Only cells with ingested organisms are represented in these results . For concentration of sera in RPMI, see text .
intracellular organisms did not increase (Table 2) and the predominant fluorescence characteristics changed to the metachromasy of dead bacteria . Discussion and conclusions In these experiments, the AO-CV technique developed by previous workers' 5,17,19,20 was modified to examine the phagocytic index and subsequent bactericidal activity of macrophages for various isolates of S . suis type 2 . The modification of removing noningested organisms after an initial period of incubation and then reincubating the cell preparations allowed the sequential phases of phagocytosis and intracellular killing to be studied separately . Preliminary studies omitting CV staining had shown that not all extracellular organisms were removed by washing after the initial 60 min incubation period ; rare extracellular bacteria (< 0 .2% total bacteria observed) were seen . However, the proportion of cells containing intracellular organisms remained constant throughout the reincubation period and the mean number of ingested non-pathogenic organisms per cell did not increase (see Table 1 ) . Thus further phagocytosis of extracellular organisms during the 90 min reincubation period did not occur to a significant degree and therefore one can conclude that any observed increase in the number of intracellular organisms during the reincubation period was due to bacterial replication . These studies showed a difference in the nature of the interaction of elicited macrophages with pathogenic and non-pathogenic isolates of S . suis type 2 in vitro ; pathogenic isolates replicated within these cells whereas ingested non-pathogenic organisms were killed . Furthermore, some macrophages containing replicating organisms showed morphological changes consistent with cellular degeneration after 6090 min of reincubation, indicating that such replication may eventually cause the destruction of the phagocyte . These findings therefore substantiate a conclusion previously drawn from an ultrastructural examination of meningeal exudate in cases of S. suis type 2 meningitis that bacteria may replicate within macrophages .' The 100% correlation between intracellular replication within macrophages reported here and pathogenicity in vivo is of particular significance because it indicates that some bacterial factor (or factors) determines the outcome of macrophage-S . suis type 2 interactions that reflect pathogenicity . It is well recognized that the level and duration of bacteraemia are major determinants in the pathogenesis of meningitis . 7-1° The most important cells responsible for clearance of circulating organisms in the non-immune animal are the macrophages of the reticulo-endothelial system (RES) in the liver and spleen .' 1-13 Thus the functional status of the RES is important in reducing the number of circulating organisms available to infect various sites (e .g . the CSF) and therefore in reducing the incidence of meningitis .' 1-13 The results of the present studies suggest that circulating pathogenic
Survival of pathogenic S . suis type 2 in macrophages
193
S. suis type 2 organisms are unlikely to be cleared by the RES in pigs lacking antibodies against S . suis type 2, and so a persistent bacteraemia will develop, favouring the development of meningitis . In contrast, circulating non-pathogenic organisms would be eliminated . The importance of the outcome of the interaction of circulating S . suis type 2 organisms with mononuclear phagocytes is dramatically demonstrated by the observation that organisms of encapsulated 'non-pathogenic' isolates-that are rapidly cleared from the circulation of mice and of pigs lacking antibodies to S . suis type 2 5 ' 14-cause meningitis when inoculated into the cisterna magnum but do not cause naturally-occurring meningitis nor meningitis when inoculated intravenously .' .'
The role of antibody in S . suis type 2-macrophage interactions The effect of specific antibody in macrophage-S. suis type 2 interactions was also investigated in the present studies . Only the role of type-specific antibody in intracellular killing of pathogenic S . suis type 2 isolates was examined because problems interpreting results obtained in preliminary experiments due to clumping of organisms in hyperimmune (rabbit) and immune (pig) sera prevented accurate determination of the role of such antibody in phagocytosis . The present studies showed that, in the presence of complement factors, anti-S . suis type 2 antibody mediated intracellular killing of phagocytosed pathogenic organisms by macrophages . Intracellular organisms were killed only in the presence of both antibody and complement factors, a result consistent with previous investigations of the interaction of S . suis type 2 with polymorphs ."' The present results therefore suggest that phagocytosed pathogenic organisms would be killed by the macrophages of the RES in pigs with anti-S . suis type 2 antibodies . Since the level and duration of bacteraemia are important factors in the pathogenesis of meningitis (see above), the presence of type-specific antibody would promote the clearance of a S . suis type 2 bacteraemia and so meningitis would be less likely to develop . It is pertinent to note here that in the present studies anti-S . suis type 2 antibody was added to the macrophage preparations after phagocytosis had occurred (after the first phase of the assay) and therefore killing of ingested P1 /7 (pathogenic) organisms was mediated by a non-opsonic mechanism of the antibody . A similar phenomenon of intracellular bactericidal activity of an antibody entering the phagocyte after ingestion of organisms had occurred has also been recorded for Group B Streptococcus type Ic . 27-30 Whilst it has been demonstrated that such antibody attaches to Fc receptors on the neutrophil surface membrane, enters the cell and penetrates into the phagolysosome to coat the bacterial surface, 29 ' 30 the mechanisms by which such antibody enters the phagolysosome and mediates killing requires further investigation .
Materials and methods
Macrophages . Elicited murine peritoneal macrophages were obtained using standard procedures ." Two, 2-4-month-old ICI mice of either sex were used in each individual assay . The mice were inoculated i .p . with 1 .5 ml of aged sterile broth [3% thioglycollate broth (Brewer) ; Oxoid] and were killed 4 days later . Macrophages were recovered from the freshly-killed mice by washing the peritoneal cavity with 1 .0 ml Ca, Mg-free HBSS, pooled, pelleted by centrifugation at 600xg for 15 min, washed (by centrifugation) three times in Ca, Mg-free HESS and finally resuspended in 2 .0 ml culture medium [RPMI 1640 with glutamine and 20 mm HEPES, lacking bicarbonate and antibiotics, and supplemented with 10% heat-inactivated foetal calf serum ; Flow Laboratories (Med/FCS)] pre-warmed to 37°C ; 0 .2 ml of the macrophage suspension was placed on each of nine 19 mm circular glass coverslips and these were incubated in a humid atmosphere for 45 min at 37°C in 5% CO 2 . Non-adherent cells were removed by two washings with Ca, Mg-free HBSS ; these washings were retained . The remaining 0.2 ml of the cell suspension was used to determine, using a Coulter counter, the number of cells in the
194
A . E . Williams Table 3 Isolate P1 /7 R75/L 1 H11/1NEW GB1 TD/10 H11 /1 OLD
Isolates of S. suis type 2 used in these studies Source Pig meningitis Human meningitis H11/1OLDb Pig meningitis Pig tonsil (asymptomatic carrier) Pig tonsil (asymptomatic carrier)
Pathogenicity'
Reference 4 4,32 6 6 5,33 6
± Pathogenicity following i .v . inoculation of 109 cfu organisms in pigs : +++, meningitis, arthritis, serositis; -, none or mild transient lameness . 'Storage of H11/1OLD in liquid blood-broth culture at 4±C for 2-3 weeks results in the development of haemolytic activity, which is maintained thereafter on repeated passage, and pathogenicity .'
suspension placed on the coverslips and to make smears to determine the proportion of macrophages in the cell suspension . To ensure reproducibility of the macrophage preparations, the number of macrophages adherent to a coverslip in an assay was estimated by determining the number of cells in 0 .2 ml of the suspension placed on each coverslip (see above) and measuring the total volume of Ca, Mg-free HBSS used to wash the coverslips and the number of cells (non-adherent cells) in the collected washings .
Bacteria . Four pathogenic isolates and two encapsulated, non-pathogenic isolates of S . suis type 2 were used in these studies ; the virulence of these isolates had been previously established by i .v . inoculation of susceptible pigs` (Table 3) . Organisms in mid-exponential phase of growth were obtained from a 7-9 h culture of S . suis type 2 in Todd-Hewitt Broth, sub-cultured from a 12 h culture in 10% horse-blood broth . Twenty millilitres of growth in Todd-Hewitt Broth was pelleted by centrifugation and resuspended in a similar volume of McCoy's 5A Salt Solution (Flow Laboratories) . An estimation of the concentration of organisms present was made using a Thoma bacterial cell counting chamber and the suspension of organisms in McCoy's 5A adjusted so that 0 .2 ml contained 10 times more live organisms the number of macrophages adherent on the coverslips in that assay [about 90% of the bacteria in a 7-9 h culture of S. suis type 2 organisms in Todd-Hewitt Broth are viable (M . R . Enright, personal communication ; A . Williams, personal observations)] . An accurate determination of the concentration of live bacteria in the final suspension was made using a pour-plate method .' Stains . Acridine orange (Sigma) was used at a concentration of 14 .4 mg/100 ml sterile Gey's Balanced Salt solution and crystal violet (Hopkin and Williams) at a concentration of 50 mg/100 ml of 0 .15M (0 .85%) sterile saline . These stains, and all other salt solutions used, were adjusted to a pH of 7 .2 using 7 .5% sodium bicarbonate and warmed to 37±C before use . Assay . Two isolates were tested in each individual assay and each isolate was tested at least three times . In each assay, four coverslips were incubated with 0 .2 ml of bacterial suspension of one isolate and four more with the other . In most cases, one pathogenic and one nonpathogenic isolate were used in each assay to ensure that the behaviour of the macrophages did not vary between assays . A ninth coverslip acted as a control for macrophage viability throughout the assay and was incubated with 0 .2 ml sterile McCoy's 5A solution . The coverslips were incubated in a humid atmosphere for 60 min at 37±C in 5% CO 2 . After this time, the coverslips were washed five times with Ca, Mg-free HBSS to remove extracellular organisms, covered with warmed Med/FCS and reincubated for a further 0, 30, 60, or 90 min in similar conditions . At each time interval, one coverslip from each of the two isolates being tested was washed three times with Ca, Mg-free HBSS, stained with acridine orange for 45 s, washed twice, stained with crystal violet, washed twice, placed cell-side downwards onto a clean glass microscope slide and sealed with nail varnish . The control macrophage preparation was stained after 90 min reincubation . The preparations were viewed under oil at a magnification of x 1000 using a Nikon Optiphot
Survival of pathogenic S. suis type 2 in macrophages
195
microscope with a mercury lamp source, a 450-490 nm excitation filter, a 510 dichroic mirror and a 520 nm barrier filter (Nikon filter cassette B 2 ) . Cells were counted until 50 had been observed to contain intracellular organisms . The proportion of cells containing ingested organisms, the number of organisms in each cell showing phagocytosis and the colour of fluorescence of ingested bacteria were recorded . Studies using rabbit sera . A further series of experiments was performed in which the foetal calf serum in the Med/FCS used during the reincubation period was replaced by fresh rabbit serum . The rabbit serum added was either : normal serum (1 : 5 in RPMI 1640), hyperimmune serum raised against washed, formalin-killed cultures of S . suis type 2 34 (1 :10) or hyperimmune serum previously heated at 56°C for 20 min to remove complement factors (1 :10) . The same pathogenic isolate (P1 /7) was used in all these studies .
The author is pleased to acknowledge the assistance of W . F . Blakemore . This work was supported by funds from the Agricultural and Food Research Council, U .K .
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