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In Vitro Demonstration of the Invasive Ability of Campylobacters
Zbl. Bakt. 283, 306-313 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York
In Vitro Demonstration of the Invasive Ability of Campylobacters SUN TEE TAY, SHAMALA DEVI, SAVITHRI PUTHUCHEARY, and INGRID KAUTNER Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Received October 17, 1994 . Revision received February 22, 1995 . Accepted July 20,1995
Summary By means of the gentamicin HEp-2 cell invasion assay, it was demonstrated that 82 % of the Campylobacters tested were cell-invasive, including 83% of isolates from bloody diarrhoea and 80% of isolates from watery diarrhoea. The large number of invasive strains from watery diarrhoea suggests the possible role of invasiveness in the production of watery diarrhoea. Whether this stage can progress further to more severe symptoms such as bloody diarrhoea remains to be elucidated. Whether this progression to bloody diarrhoea occurs as a result of toxin production is still debatable. In Vero cells, invasion was less efficient and intracellular multiplication was not observed.
Introduction Bacterial infection is a complex and multifactorial phenomenon which depends on the interaction of properties belonging to both the host and the bacteria involved (8, 15). The mechanism(s) for induction of diarrhoea caused by enteropathogens include invasive ability, the production of toxins as well as others (which are not well known), but the exact mechanism(s) by which C. jejuni and C. coli bring about diarrhoea is (are) not known (4). Blood and leukocytes have frequently been observed in the faeces of patients infected with C. jejuni (2) suggesting an invasion of the intestinal mucosa. The adherence of microorganisms to mucosal surfaces is a primary step in the pathogenesis of many intestinal infections and a prerequisite for invasion. The invasive process encompasses features that include entry of the bacteria into epithelial cells, intracellular multiplication, intra- as well as intercellular spread and killing of host cells (12). At present, the interaction of campylobacters with intestinal cells is not well understood. The objective of this study was to invstigate the adherence and invasive ability of campylobacters using cultured cells of mammalian origin.
In vitro Invasive Ability of Campylobaeters
307
Materials and Methods
Bacterial isolates. Twenty-nine Campylobaeter isolates (23 clinical ones and 6 from poultry) and two reference strains (C. coli and C. lari) were tested for invasiveness in mammalian cell cultures. A clinical isolate of S. typhi and a noninvasive E. coli K12 strain were used as positive and negative controls, respectively. Gentamicin HEp-2 cell invasion assay. This method was adapted from Konkel and Joens (11). HEp-2 (human laryngeal epidermoid carcinoma. ATCC CCL 23) cells were maintained in Eagle's Minimal Essential Medium (EMEM) supplemented with 10% foetal bovine serum (FBS) without antibiotics at 37°C. Confluent cultures were trypsinized, the cells were counted and suspended in growth medium at a concentration of 3 X 10 10 per mL. A twenty-four well tissue culture tray (Costar, USA) was seeded with 0.5 mL of this cell suspension per well and incubated at 37°C for 18 h to establish confluent monolayers. The monolayers were washed and incubated with maintenance medium before use. Bacteria were harvested from blood agar plates after 24 h of incubation and suspended in maintenance medium to yield a concentration of approximately 10 8 organisms/mL. The viable count was determined by serial dilution of this suspension and inoculation of the diluted cells onto Mueller-Hinton (MH) agar plates. A volume of 0.5 mL of the bacterial suspension was inoculated into triplicate wells containing confluent monolayers of HEp-2 cells. Infected monolayers were incubated at 3rc for 3 h to allow the bacteria to adhere to the cells. The monolayers were then washed five times with PBS, pH 7.2, and reincubated for 3 h under the same conditions to allow bacterial invasion of the HEp-2 cells. Medium containing 250 llg/mL of gentamicin (Servigenta 80, Switzerland) was added to two of the wells to kill extracellular bacteria for the enumeration of intracellular bacteria. Medium without any gentamicin was added to the third well for the enumeration of both intra- and extracellular bacteria. Following incubation, the monolayers were washed three times with PBS and lysed with 0.5% sodium deoxycholate to release the bacteria. Serial dilutions of the suspensions were made and inoculated onto MH agar plates to determine the viable counts. For the third well, 0.5 mL of 2 % formaldehyde in PBS was added at the end of the experiment to fix the monolayers after which they were Giemsa-stained to determine the integrity of the monolayers. The criteria used to assess the invasive properties of campylobacters were i) visualization of intracellular bacteria in Giemsa-stained monolayers by light microscopy and ii) quantitation of intracellular bacteria by viable counts after the infected cells had been lysed with 0.5% sodium deoxycholate. The percentage of bacteria associated with the monolayer was determined by the number of CFU recovered from the lysed cells without gentamicin treatment as compared to the original inoculum. These bacteria were adherent or invasive as nonadherent bacteria had been removed by the vigorous washing 3 h after infection. % adherence =
(no. intracellular
+
adherent bacteria)/mL
no. of bacteria in inoculum/mL
X 100%
The number of bacteria recovered after an incubation of 3 h and a further 3 h of gentamicin treatment was compared with the number of bacteria initially added to the monolayer, to determine the efficiency with which bacteria became internalized within HEp-2 cells. This was expressed as the percentage of CFU recovered from the lysed monolayer after gentamicin treatment as compared to the original inoculum. The percentage entry of the invasive isolates should be greater than the percentage entry of the noninvasive E. coli control strain. no. intracellular bacteria/mL % entry = - - - - - - - - - - - X 100% no. bacteria in inoculum/mL
308
S. T. Tay, S. Devi, S. Puthucheary, and I. Kautner
Invasiveness of Campylobacter in Vera cells. The ability of campylobacters to adhere to and invade Vero (African green monkey kidney cells, ATCC CCL 81) cells was also determined. Vero cells were grown and maintained as described for HEp-2 cells, and the experimental details were as described above. Intracellular multiplication of Campylabacter. Since extracellular bacteria are not viable in the presence of gentamicin, any increase in the bacterial numbers recovered from infected monolayers must be seen as a result of the invasion and intracellular multiplication of the bacteria. For studies of intracellular multiplication, the assays were performed as described above, except that the mono layers were further incubated for 1, 2, 3 and 4 h, respectively, after gentamicin treatment. Entry phenotype. The entry phenotype was determined based on the percentage entry of the bacteria. E. coli K-12 was used as a negative control with a percentage entry of 0.0000039. A strain was considered as invasive provided the percentage entry was > 0.0000039.
Results Giemsa-stained HEp-2 cells. The mono layers remained intact after the gentamicin HEp-2 cell invasion assay at 6 h postinfection (Figs.1a and 1b). Giemsa-stained HEp-2 cells revealed the presence of numerous extracellular bacteria 6 h postinfection adhering to the cell membrane, as well as intracellular bacteria which often appeared as spirals consisting of 3-6 joined bacteria sometimes obliterating the cytoplasm (Fig. 1b). Rounding of the HEp-2 cells and vacuoles were also observed 6 h postinfection (Fig. 1c).
Fig. la. Monolayer of control HEp-2 cells (Giemsa-stained; X400).
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309
Fig. lb. Monolayer of HEp-2 cells 6 h postinfection with an invasive Campy/abaeter test isolate (Giemsa-stained; x400).
Fig.lc. Vacuoles (indicated by~) were observed in HEp-2 cells 6h pastinfection (Giemsastained; X 1000).
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S. T. Tay, S. Devi, S. Puthucheary, and I. Kautner
Invasive ability of Campylobacter in HEp-2 and Vera cells. S. typhi were used as positive control. The percentage of adherence to HEp-2 cells was found to be 32% with 0.0089% being internalized at 6h postinfecton (Table 1). The negative control, E. coli K-12, had a low percentage of internalization (0.0000039%) although 21 % of the cells of the original inoculum were found to adhere to HEp-2 cells. The C. coli reference strain was internalized within HEp-2 cells at a rate comparable to that of S. ty-
Table 1. Adherence and invasiveness of clinical, control and poultry strains of Campylobacter to HEp-2 cells Strain number
Type of stool
Percent internalization
Percent adherence
Entry phenotype
24 88 71 68 5 30 49 40
no diarrh. watery watery watery bloody bloody bloody bloody
0.09 0.0057
0.3 2.4 0.0015 3.2 1.1 3.6 0.044 0.0000037
+ +
C. jejuni biotype II
26 44 91 18 74 32 28
watery watery bloody bloody bloody bloody bloody
0.0067 0.0034 0.02 0.0024 0.Q11 0.00016 0.000079
0.29 0.008 0.031 0.021 0.054 0.0092 0.033
+ + + + + + +
C. coli
19 M23 66 37
watery watery bloody bloody
0.0012 0.0015 0.00053
0.0067 0.027 0.013
+ + +
Untypable
15 27 36
watery watery bloody
0.0098
0.0013
+
0.00028
0.0038
+
0.0089 0.0000039 0.0053
32 21 0.82 0.000045
+
Strain Clinical C. jejuni biotype I
Control
Poultry
S. typhi E. coli K 12 C. coli C. lari llR C14 16R 21R lOR Cll
0.000021 0.00028 0.26 0.025
0.00078 0.0015 0.0044 0.0091
0.000006 0.0013 0.014 0.045 0.007 0.03
+ + + +
+
+ + + +
In vitro Invasive Ability of Campylobaeters
311
phi, but the C. lari reference strain was not able to adhere to and invade HEp-2 cells as no intracellular bacteria could be recovered 6 h post-infection (Table 1). The 23 clinical and 6 poultry isolates (Table 1) demonstrated differences in the ability to adhere to and invade HEp-2 cells. Twenty-five of the isolates (86%) showed percentages of adherence ranging from 0.0013% to 3.6%. Four isolates, including three clinical and one poultry strain, adhered only very weakly or not at all (strain 40, 0.0000037%; 11R, 0.000006%; 37 and 27,0%). Two C. jejuni biotype I isolates demonstrated the highest percentages of internalization: strain 30 isolated from a bloody stool with 0.26% and strain 24 from a patient with fever but no diarrhoea with 0.09%. No correlation could be established between biotypes of C. jejuni and invasive ability. Almost all biotypes were invasive. These included 7 out of 9 (83%) C. jejuni biotype I, all 7 biotype II isolates, 3 out of 4 (75%) C. coli isolates and 2 out of 3 (68%) untypable strains. Four out of six (68 %) poultry isolates tested were found to be invasive, with percentages of internalization ranging from 0.000078% to 0.0091 % (Table 1). All Campylabaeter isolates tested were able to adhere to or invade both types of mammalian cells, regardless of their origin. However, the isolates adhered better to Vero cells, with the poultry isolate showing the highest percentage of adherence. The C. lari reference strain was unable to adhere to both the HEp-2 and Vero cells. Although the clinical isolates adhered better to Vero cells, their percentages of internalization were much higher in HEp-2 cells. There was no difference in the invasive ability of strains isolated from watery or bloody stools (data not shown). Intracellular multiplication of eampylabaeters. There was a slight increase in the number of bacteria 3-4 h postinfection. After further incubation, the number of viable bacteria recovered dropped sharply for all isolates tested (data not shown). Therefore, in general, the numbers of CFU of the invasive Campylabaeter isolates were reduced after prolonged incubation. The S. typhi control strain showed a slight increase in the CFU count during incubation.
Discussion Various studies suggest that enteropathogens have invasive properties (14, 8). The ability to adhere to and invade epithelial cells is considered to be a prerequisite for virulence. Invasion of epithelial cells probably contributes to the systemic symptoms seen in some enteric infections. The gentamicin HEp-2 cell invasion assay used in this study is a simple, reproducible and quantitative in vitro test and has been shown to correlate well with in vivo bacterial invasion of intestinal epithelium (6). Gentamicin is a bactericidal antibiotic that is unable to enter eukaryotic cells. Therefore, bacteria adhering to epithelial cells are killed by this drug, whereas internalized bacteria are protected from its effects (7, 9, 11). This study has shown that 83% of the Campylabaeter clinical isolates were able to invade HEp-2 cells. Similarly, Konkel and Joens (11) showed that 19 of 21 (91 %) isolates from patients with clinical signs of Campylabaeter infection were invasive. In our study, there was no significant correlation between biotypes and invasive ability of Campylabaeter isolates (Table 1). The ability of Campylabaeter isolates to enter epithelial cells varied considerably between strains. The percentage of internalization in this study ranged from 0.000021 % to 0.26% for clinical isolates (Table 1). Since it has been demonstrated that mixed infections of campylobacters with other enteropathogens facilitated the in-
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Table 2. The invasive ability of strains isolated from watery or bloody stools Clinical manifestations
Entry phenotye Total
Watery stools Bloody stools
8 (80%) 10 (83%)
2 (20%) 2 (17%)
12
Total
18 (82%)
4 (18%)
22
10
vasiveness of campylobacters in mammalian cell culture models (3), the invasive ability of campylobacters in vivo could be greater. In fact, isolation of campylobacters together with other enteropathogens in clinical samples or polymicrobial infections is common (13, 16). The invasive ability of poultry isolates as seen in four out of six strains in this study (Table 1) suggests the potential role of poultry strains in causing human Campylobaeter infections. In this study, 83% of the isolates from bloody diarrhoea and 80% of the isolates from watery diarrhoea were found to be invasive (Table 2). Although invasiveness has been reported to be consistent with bloody diarrhoea and inflammatory lesions (10), the large number of invasive isolates from watery diarrhoea suggests the possible role of invasiveness in the production of watery diarrhoea. In vivo experiments have demonstrated that invasive strains could induce fluid secretion in the ligated ileal loop of the rat (10) although the precise mechanism by which the invasive strains causes fluid secretion in this animal model is obscure. Whether this could result in progression to bloody diarrhoea with the productin of toxins is debatable. A temporary loss of brush border function may occur after Campylobaeter invasion of the mucosal epithelium which causes the induction of malabsorptive and watery diarrhoea without discernible villus atrophy (1). The ability to adhere to and invade human and animal cell lines indicated that cell invasion is not limited to one epithelial cell type. The different rates of internalization of bacteria observed in different cell lines suggest that the host cells play important roles in the invasion process. In this study, the type and density of receptors on the cell surface of mammalian cells could determine the efficiency of bacterial internalization. The CFU count of organisms recovered from the infected mono layers gradually decreased after extended periods of incubation. This is in contrast to the behaviour of S. typhi clinical strains and Shigella, Salmonella and EIEC which have been shown to display rapid intracellular multiplication (6, 8). The failure of campylobacters to multiply intracellularly could be due to the susceptibility of the organism to host killing mechanisms. Ingested campylobacters have been demonstrated to remain enveloped within endocytic vacuoles in the cytoplasm of the host cell. The occurrence of phagolysosome fusion could be correlated to the decrease in viable internalized bacteria observed after 9 hours of incubation (5). In this study, vacuoles were observed in the epithelial cells 6 hours postinfeetion (Fig. Ie). The ability to enter epithelial cells is considered a prerequisite for virulence. Intracellular organisms, however, may be able to disrupt the cellular processes and therefore interfere with cell architecture. In addition, organisms secreting low levels of cy-
In vitro Invasive Ability of Campylobaeters
313
totoxins may be more virulent once they are in an intracellular location. However, as shown in many studies, invasiveness is not the only virulence factor responsible for the pathogenicity of campylobacters.
Acknowledgements. This work forms part of the M. Med. Sci. thesis of the first author and was supported by the University of Malaya and the Ministry of Science, Technology and Environment, Malaysia (R & D) grant ## 3/077/01. References 1. Bell, J. A. and D. D. Manning: Role of temporary intestinal brush border dysfunction in Campylobaeter. Curro Microbio!. 21 (1990) 355-359 2. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M.J. Blaser: Experimental Campylobaeter jejuni infections in humans. J. Infect. Dis. 157 (1988) 472-479 3. Bukholm, G. and G. Kapperud: Expression of Campylobaeter jejuni invasiveness in cell cultures coinfected with other bacteria. Infect. Immun. 55 (1987) 2816-2821 4. Butzler, J.-P., Y. Glupezynski, and H. Goossens: Campylobaeter and Helicobaeter infections. Curro Opinion in Infectious Diseases 5 (1992) 80-87 5. DeMelo, M. A., G. Gabbiani, andJ.-C. Peehere: Cellular events and intracellular survival of Campylobaeter jejuni during infection of HEp-2 cells. Infect. Immun. 57 (1989) 2214-2222 6. Donnenberg, M. S., A. Donohue-Rolfe, and G. T. Keuseh: Epithelial cell invasion: An overlooked property of enteropathogenic Escherichia coli (EPEG) associated with the EPEC adherence factor. J. Infect. Dis. 160 (1989) 452-459 7. Falkow, S., P. Small, R. Isenberg, S. F. Hayes, and D. Corwin: A molecular strategy for the study of bacterial invasion. Rev. Infect. Dis. 9 (1987) 5450-5455 8. Finlay, H. B. and S. Falkow: Common themes in microbial pathogenicity. Microbio!. Re~53
(1989)210-230
9. Geme III, J. W. St. and S. Falkow: Haemophilus in(luenzae adheres to and enters cultured human epithelial cells. Infect. Immun. 58 (1990) 4036-4044
10. Klipstein, F. A., R. F. Engert, and H. B. Short: Enzyme-linked immunosorbent assays for
virulence properties of Campylobaeter jejuni clinical isolates. J. Clin. Microbiol. 23 (1986) 1039-1043 11. Konkel, M. E. and L. A. Joens: Adhesion to and invasion of HEp-2 cells by Campylobaeter spp. Infect. Immun. 57 (1989) 2984-2990 12. Maurelli, A. T. and P.]. Sansonetti: Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc. Nat!' Acad. Sci. U.S.A. 85 (1988) 2820-2824 13. Ruiz-Palaeios, G. M., Y. Lopez-Vidal, ]. Torres, and N. Torres: Serum antibodies to heat-labile enterotoxin of Campylobaeter jejuni. J. Infect. Dis. 152 (1985) 413-416 14. Small, P. L. c., R. R. Isenberg, and S. Falkow: Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enteroeolitiea to enter and replicate within HEp-2 cells. Infect. Immun. 55 (1987) 1674-1679 15. Smith, H.: The biochemical challenge of microbial pathogenicity. App!. Bacteriol. 57 (1984) 395-404 16. Taylor, D. N., P. Eehieverria, M.]. Blaser, C. Pitarangsi, N. Blaeklow,]. Cross, and B. G. Weniger: Polymicrobial aetiology of travellers' diarrhoea. Lancet i (1985) 381-383 Prof. Savithri Puthuehaery, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia
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Zhl. Bakt. 283/3
In Vitro Demonstration of the Invasive Ability of Campylobacters SUN TEE TAY, SHAMALA DEVI, SAVITHRI PUTHUCHEARY, and INGRID KAUTNER Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Received October 17, 1994 . Revision received February 22, 1995 . Accepted July 20,1995
Summary By means of the gentamicin HEp-2 cell invasion assay, it was demonstrated that 82 % of the Campylobacters tested were cell-invasive, including 83% of isolates from bloody diarrhoea and 80% of isolates from watery diarrhoea. The large number of invasive strains from watery diarrhoea suggests the possible role of invasiveness in the production of watery diarrhoea. Whether this stage can progress further to more severe symptoms such as bloody diarrhoea remains to be elucidated. Whether this progression to bloody diarrhoea occurs as a result of toxin production is still debatable. In Vero cells, invasion was less efficient and intracellular multiplication was not observed.
Introduction Bacterial infection is a complex and multifactorial phenomenon which depends on the interaction of properties belonging to both the host and the bacteria involved (8, 15). The mechanism(s) for induction of diarrhoea caused by enteropathogens include invasive ability, the production of toxins as well as others (which are not well known), but the exact mechanism(s) by which C. jejuni and C. coli bring about diarrhoea is (are) not known (4). Blood and leukocytes have frequently been observed in the faeces of patients infected with C. jejuni (2) suggesting an invasion of the intestinal mucosa. The adherence of microorganisms to mucosal surfaces is a primary step in the pathogenesis of many intestinal infections and a prerequisite for invasion. The invasive process encompasses features that include entry of the bacteria into epithelial cells, intracellular multiplication, intra- as well as intercellular spread and killing of host cells (12). At present, the interaction of campylobacters with intestinal cells is not well understood. The objective of this study was to invstigate the adherence and invasive ability of campylobacters using cultured cells of mammalian origin.
In vitro Invasive Ability of Campylobaeters
307
Materials and Methods
Bacterial isolates. Twenty-nine Campylobaeter isolates (23 clinical ones and 6 from poultry) and two reference strains (C. coli and C. lari) were tested for invasiveness in mammalian cell cultures. A clinical isolate of S. typhi and a noninvasive E. coli K12 strain were used as positive and negative controls, respectively. Gentamicin HEp-2 cell invasion assay. This method was adapted from Konkel and Joens (11). HEp-2 (human laryngeal epidermoid carcinoma. ATCC CCL 23) cells were maintained in Eagle's Minimal Essential Medium (EMEM) supplemented with 10% foetal bovine serum (FBS) without antibiotics at 37°C. Confluent cultures were trypsinized, the cells were counted and suspended in growth medium at a concentration of 3 X 10 10 per mL. A twenty-four well tissue culture tray (Costar, USA) was seeded with 0.5 mL of this cell suspension per well and incubated at 37°C for 18 h to establish confluent monolayers. The monolayers were washed and incubated with maintenance medium before use. Bacteria were harvested from blood agar plates after 24 h of incubation and suspended in maintenance medium to yield a concentration of approximately 10 8 organisms/mL. The viable count was determined by serial dilution of this suspension and inoculation of the diluted cells onto Mueller-Hinton (MH) agar plates. A volume of 0.5 mL of the bacterial suspension was inoculated into triplicate wells containing confluent monolayers of HEp-2 cells. Infected monolayers were incubated at 3rc for 3 h to allow the bacteria to adhere to the cells. The monolayers were then washed five times with PBS, pH 7.2, and reincubated for 3 h under the same conditions to allow bacterial invasion of the HEp-2 cells. Medium containing 250 llg/mL of gentamicin (Servigenta 80, Switzerland) was added to two of the wells to kill extracellular bacteria for the enumeration of intracellular bacteria. Medium without any gentamicin was added to the third well for the enumeration of both intra- and extracellular bacteria. Following incubation, the monolayers were washed three times with PBS and lysed with 0.5% sodium deoxycholate to release the bacteria. Serial dilutions of the suspensions were made and inoculated onto MH agar plates to determine the viable counts. For the third well, 0.5 mL of 2 % formaldehyde in PBS was added at the end of the experiment to fix the monolayers after which they were Giemsa-stained to determine the integrity of the monolayers. The criteria used to assess the invasive properties of campylobacters were i) visualization of intracellular bacteria in Giemsa-stained monolayers by light microscopy and ii) quantitation of intracellular bacteria by viable counts after the infected cells had been lysed with 0.5% sodium deoxycholate. The percentage of bacteria associated with the monolayer was determined by the number of CFU recovered from the lysed cells without gentamicin treatment as compared to the original inoculum. These bacteria were adherent or invasive as nonadherent bacteria had been removed by the vigorous washing 3 h after infection. % adherence =
(no. intracellular
+
adherent bacteria)/mL
no. of bacteria in inoculum/mL
X 100%
The number of bacteria recovered after an incubation of 3 h and a further 3 h of gentamicin treatment was compared with the number of bacteria initially added to the monolayer, to determine the efficiency with which bacteria became internalized within HEp-2 cells. This was expressed as the percentage of CFU recovered from the lysed monolayer after gentamicin treatment as compared to the original inoculum. The percentage entry of the invasive isolates should be greater than the percentage entry of the noninvasive E. coli control strain. no. intracellular bacteria/mL % entry = - - - - - - - - - - - X 100% no. bacteria in inoculum/mL
308
S. T. Tay, S. Devi, S. Puthucheary, and I. Kautner
Invasiveness of Campylobacter in Vera cells. The ability of campylobacters to adhere to and invade Vero (African green monkey kidney cells, ATCC CCL 81) cells was also determined. Vero cells were grown and maintained as described for HEp-2 cells, and the experimental details were as described above. Intracellular multiplication of Campylabacter. Since extracellular bacteria are not viable in the presence of gentamicin, any increase in the bacterial numbers recovered from infected monolayers must be seen as a result of the invasion and intracellular multiplication of the bacteria. For studies of intracellular multiplication, the assays were performed as described above, except that the mono layers were further incubated for 1, 2, 3 and 4 h, respectively, after gentamicin treatment. Entry phenotype. The entry phenotype was determined based on the percentage entry of the bacteria. E. coli K-12 was used as a negative control with a percentage entry of 0.0000039. A strain was considered as invasive provided the percentage entry was > 0.0000039.
Results Giemsa-stained HEp-2 cells. The mono layers remained intact after the gentamicin HEp-2 cell invasion assay at 6 h postinfection (Figs.1a and 1b). Giemsa-stained HEp-2 cells revealed the presence of numerous extracellular bacteria 6 h postinfection adhering to the cell membrane, as well as intracellular bacteria which often appeared as spirals consisting of 3-6 joined bacteria sometimes obliterating the cytoplasm (Fig. 1b). Rounding of the HEp-2 cells and vacuoles were also observed 6 h postinfection (Fig. 1c).
Fig. la. Monolayer of control HEp-2 cells (Giemsa-stained; X400).
In vitro Invasive Ability of Campy/obaeters
309
Fig. lb. Monolayer of HEp-2 cells 6 h postinfection with an invasive Campy/abaeter test isolate (Giemsa-stained; x400).
Fig.lc. Vacuoles (indicated by~) were observed in HEp-2 cells 6h pastinfection (Giemsastained; X 1000).
310
S. T. Tay, S. Devi, S. Puthucheary, and I. Kautner
Invasive ability of Campylobacter in HEp-2 and Vera cells. S. typhi were used as positive control. The percentage of adherence to HEp-2 cells was found to be 32% with 0.0089% being internalized at 6h postinfecton (Table 1). The negative control, E. coli K-12, had a low percentage of internalization (0.0000039%) although 21 % of the cells of the original inoculum were found to adhere to HEp-2 cells. The C. coli reference strain was internalized within HEp-2 cells at a rate comparable to that of S. ty-
Table 1. Adherence and invasiveness of clinical, control and poultry strains of Campylobacter to HEp-2 cells Strain number
Type of stool
Percent internalization
Percent adherence
Entry phenotype
24 88 71 68 5 30 49 40
no diarrh. watery watery watery bloody bloody bloody bloody
0.09 0.0057
0.3 2.4 0.0015 3.2 1.1 3.6 0.044 0.0000037
+ +
C. jejuni biotype II
26 44 91 18 74 32 28
watery watery bloody bloody bloody bloody bloody
0.0067 0.0034 0.02 0.0024 0.Q11 0.00016 0.000079
0.29 0.008 0.031 0.021 0.054 0.0092 0.033
+ + + + + + +
C. coli
19 M23 66 37
watery watery bloody bloody
0.0012 0.0015 0.00053
0.0067 0.027 0.013
+ + +
Untypable
15 27 36
watery watery bloody
0.0098
0.0013
+
0.00028
0.0038
+
0.0089 0.0000039 0.0053
32 21 0.82 0.000045
+
Strain Clinical C. jejuni biotype I
Control
Poultry
S. typhi E. coli K 12 C. coli C. lari llR C14 16R 21R lOR Cll
0.000021 0.00028 0.26 0.025
0.00078 0.0015 0.0044 0.0091
0.000006 0.0013 0.014 0.045 0.007 0.03
+ + + +
+
+ + + +
In vitro Invasive Ability of Campylobaeters
311
phi, but the C. lari reference strain was not able to adhere to and invade HEp-2 cells as no intracellular bacteria could be recovered 6 h post-infection (Table 1). The 23 clinical and 6 poultry isolates (Table 1) demonstrated differences in the ability to adhere to and invade HEp-2 cells. Twenty-five of the isolates (86%) showed percentages of adherence ranging from 0.0013% to 3.6%. Four isolates, including three clinical and one poultry strain, adhered only very weakly or not at all (strain 40, 0.0000037%; 11R, 0.000006%; 37 and 27,0%). Two C. jejuni biotype I isolates demonstrated the highest percentages of internalization: strain 30 isolated from a bloody stool with 0.26% and strain 24 from a patient with fever but no diarrhoea with 0.09%. No correlation could be established between biotypes of C. jejuni and invasive ability. Almost all biotypes were invasive. These included 7 out of 9 (83%) C. jejuni biotype I, all 7 biotype II isolates, 3 out of 4 (75%) C. coli isolates and 2 out of 3 (68%) untypable strains. Four out of six (68 %) poultry isolates tested were found to be invasive, with percentages of internalization ranging from 0.000078% to 0.0091 % (Table 1). All Campylabaeter isolates tested were able to adhere to or invade both types of mammalian cells, regardless of their origin. However, the isolates adhered better to Vero cells, with the poultry isolate showing the highest percentage of adherence. The C. lari reference strain was unable to adhere to both the HEp-2 and Vero cells. Although the clinical isolates adhered better to Vero cells, their percentages of internalization were much higher in HEp-2 cells. There was no difference in the invasive ability of strains isolated from watery or bloody stools (data not shown). Intracellular multiplication of eampylabaeters. There was a slight increase in the number of bacteria 3-4 h postinfection. After further incubation, the number of viable bacteria recovered dropped sharply for all isolates tested (data not shown). Therefore, in general, the numbers of CFU of the invasive Campylabaeter isolates were reduced after prolonged incubation. The S. typhi control strain showed a slight increase in the CFU count during incubation.
Discussion Various studies suggest that enteropathogens have invasive properties (14, 8). The ability to adhere to and invade epithelial cells is considered to be a prerequisite for virulence. Invasion of epithelial cells probably contributes to the systemic symptoms seen in some enteric infections. The gentamicin HEp-2 cell invasion assay used in this study is a simple, reproducible and quantitative in vitro test and has been shown to correlate well with in vivo bacterial invasion of intestinal epithelium (6). Gentamicin is a bactericidal antibiotic that is unable to enter eukaryotic cells. Therefore, bacteria adhering to epithelial cells are killed by this drug, whereas internalized bacteria are protected from its effects (7, 9, 11). This study has shown that 83% of the Campylabaeter clinical isolates were able to invade HEp-2 cells. Similarly, Konkel and Joens (11) showed that 19 of 21 (91 %) isolates from patients with clinical signs of Campylabaeter infection were invasive. In our study, there was no significant correlation between biotypes and invasive ability of Campylabaeter isolates (Table 1). The ability of Campylabaeter isolates to enter epithelial cells varied considerably between strains. The percentage of internalization in this study ranged from 0.000021 % to 0.26% for clinical isolates (Table 1). Since it has been demonstrated that mixed infections of campylobacters with other enteropathogens facilitated the in-
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S. T. Tay, S. Devi, S. Puthucheary, and I. Kautner
Table 2. The invasive ability of strains isolated from watery or bloody stools Clinical manifestations
Entry phenotye Total
Watery stools Bloody stools
8 (80%) 10 (83%)
2 (20%) 2 (17%)
12
Total
18 (82%)
4 (18%)
22
10
vasiveness of campylobacters in mammalian cell culture models (3), the invasive ability of campylobacters in vivo could be greater. In fact, isolation of campylobacters together with other enteropathogens in clinical samples or polymicrobial infections is common (13, 16). The invasive ability of poultry isolates as seen in four out of six strains in this study (Table 1) suggests the potential role of poultry strains in causing human Campylobaeter infections. In this study, 83% of the isolates from bloody diarrhoea and 80% of the isolates from watery diarrhoea were found to be invasive (Table 2). Although invasiveness has been reported to be consistent with bloody diarrhoea and inflammatory lesions (10), the large number of invasive isolates from watery diarrhoea suggests the possible role of invasiveness in the production of watery diarrhoea. In vivo experiments have demonstrated that invasive strains could induce fluid secretion in the ligated ileal loop of the rat (10) although the precise mechanism by which the invasive strains causes fluid secretion in this animal model is obscure. Whether this could result in progression to bloody diarrhoea with the productin of toxins is debatable. A temporary loss of brush border function may occur after Campylobaeter invasion of the mucosal epithelium which causes the induction of malabsorptive and watery diarrhoea without discernible villus atrophy (1). The ability to adhere to and invade human and animal cell lines indicated that cell invasion is not limited to one epithelial cell type. The different rates of internalization of bacteria observed in different cell lines suggest that the host cells play important roles in the invasion process. In this study, the type and density of receptors on the cell surface of mammalian cells could determine the efficiency of bacterial internalization. The CFU count of organisms recovered from the infected mono layers gradually decreased after extended periods of incubation. This is in contrast to the behaviour of S. typhi clinical strains and Shigella, Salmonella and EIEC which have been shown to display rapid intracellular multiplication (6, 8). The failure of campylobacters to multiply intracellularly could be due to the susceptibility of the organism to host killing mechanisms. Ingested campylobacters have been demonstrated to remain enveloped within endocytic vacuoles in the cytoplasm of the host cell. The occurrence of phagolysosome fusion could be correlated to the decrease in viable internalized bacteria observed after 9 hours of incubation (5). In this study, vacuoles were observed in the epithelial cells 6 hours postinfeetion (Fig. Ie). The ability to enter epithelial cells is considered a prerequisite for virulence. Intracellular organisms, however, may be able to disrupt the cellular processes and therefore interfere with cell architecture. In addition, organisms secreting low levels of cy-
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313
totoxins may be more virulent once they are in an intracellular location. However, as shown in many studies, invasiveness is not the only virulence factor responsible for the pathogenicity of campylobacters.
Acknowledgements. This work forms part of the M. Med. Sci. thesis of the first author and was supported by the University of Malaya and the Ministry of Science, Technology and Environment, Malaysia (R & D) grant ## 3/077/01. References 1. Bell, J. A. and D. D. Manning: Role of temporary intestinal brush border dysfunction in Campylobaeter. Curro Microbio!. 21 (1990) 355-359 2. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M.J. Blaser: Experimental Campylobaeter jejuni infections in humans. J. Infect. Dis. 157 (1988) 472-479 3. Bukholm, G. and G. Kapperud: Expression of Campylobaeter jejuni invasiveness in cell cultures coinfected with other bacteria. Infect. Immun. 55 (1987) 2816-2821 4. Butzler, J.-P., Y. Glupezynski, and H. Goossens: Campylobaeter and Helicobaeter infections. Curro Opinion in Infectious Diseases 5 (1992) 80-87 5. DeMelo, M. A., G. Gabbiani, andJ.-C. Peehere: Cellular events and intracellular survival of Campylobaeter jejuni during infection of HEp-2 cells. Infect. Immun. 57 (1989) 2214-2222 6. Donnenberg, M. S., A. Donohue-Rolfe, and G. T. Keuseh: Epithelial cell invasion: An overlooked property of enteropathogenic Escherichia coli (EPEG) associated with the EPEC adherence factor. J. Infect. Dis. 160 (1989) 452-459 7. Falkow, S., P. Small, R. Isenberg, S. F. Hayes, and D. Corwin: A molecular strategy for the study of bacterial invasion. Rev. Infect. Dis. 9 (1987) 5450-5455 8. Finlay, H. B. and S. Falkow: Common themes in microbial pathogenicity. Microbio!. Re~53
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9. Geme III, J. W. St. and S. Falkow: Haemophilus in(luenzae adheres to and enters cultured human epithelial cells. Infect. Immun. 58 (1990) 4036-4044
10. Klipstein, F. A., R. F. Engert, and H. B. Short: Enzyme-linked immunosorbent assays for
virulence properties of Campylobaeter jejuni clinical isolates. J. Clin. Microbiol. 23 (1986) 1039-1043 11. Konkel, M. E. and L. A. Joens: Adhesion to and invasion of HEp-2 cells by Campylobaeter spp. Infect. Immun. 57 (1989) 2984-2990 12. Maurelli, A. T. and P.]. Sansonetti: Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc. Nat!' Acad. Sci. U.S.A. 85 (1988) 2820-2824 13. Ruiz-Palaeios, G. M., Y. Lopez-Vidal, ]. Torres, and N. Torres: Serum antibodies to heat-labile enterotoxin of Campylobaeter jejuni. J. Infect. Dis. 152 (1985) 413-416 14. Small, P. L. c., R. R. Isenberg, and S. Falkow: Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enteroeolitiea to enter and replicate within HEp-2 cells. Infect. Immun. 55 (1987) 1674-1679 15. Smith, H.: The biochemical challenge of microbial pathogenicity. App!. Bacteriol. 57 (1984) 395-404 16. Taylor, D. N., P. Eehieverria, M.]. Blaser, C. Pitarangsi, N. Blaeklow,]. Cross, and B. G. Weniger: Polymicrobial aetiology of travellers' diarrhoea. Lancet i (1985) 381-383 Prof. Savithri Puthuehaery, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur, Malaysia
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