MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 92(4) Jul/Ago 1997
PAGES: 509-511 DOI: Technical notes
Detection of Campylobacter jejuni Invasion of HEp-2 Cells by Acridine Orange-Crystal Violet Staining

H Fernández +, G EllerI, E FreymullerII, T Vivanco

Instituto de Microbiología Clinica 
IInstituto de Inmunologia, Universidad Austral de Chile, Valdivia, Chile 
IICentro de Microscopia Eletrônica, Universidade Federal de São Paulo, São Paulo, SP, Brasil

RESEARCH NOTE

Since the identification of Campylobacter jejuni as an enteropathogenic agent, it has been shown that the clinical picture of Campylobacter enteritis suggests the existence of an invasive mechanism in this bacteria (R Walker et al. 1986 Microbiol Rev 50: 81-94). The ability of C. jejuni to invade epithelial cells was confirmed in vivo using chicken (L Field et al. 1986 Infect Immun 54: 118-125), mice (M Youssef et al. 1987 Infect Immun 55: 1019-1021), hamsters (C Humphrey et al. 1985 J Infect Dis 152: 485-493), and monkeys (R Russel et al. 1989 Infect Immun 57: 1438-1444) as biological models. Their in vitro invasion potential has been demonstrated in mammalian cell lines using electron microscopy (M de Melo et al. 1989 Infect Immun 57: 2214-2222), indirect immunofluorescence techniques (M Konkel & L Joens 1989 Infect Immun 57: 2984-2990), radiolabelled bacteria (G Lindblom et al. 1990 APMIS 98: 179-184) and May Grünwald Giemsa staining (H Fernández & L Trabulsi 1995 Biol Res 28: 205-210). Most of the latter techniques are cumbersome or difficult to be carried out or even difficult to read and interpret. Recently, the use of an acridine orange staining technique combined with crystal violet counterstaining (AO-CV stain) was proposed to investigate the presence of several intracellular enteropathogens other than Campylobacter in HeLa cells (M Milliotis 1991 J Clin Microbiol 29: 830-831).

In the present study we used this technique to demonstrate invasiveness of C. jejuni, by employing transmission electron microscopy (TEM) as standard control.

Twenty C. jejuni strains were tested for in vitro invasiveness. The source of the strains is shown in TableIn vitro invasiveness tests were carried out as previously described (Fernández & Trabulsi loc. cit.) but using AO-CV stain (Milliotis loc. cit.). In brief, HEp-2 cells were cultured overnight on coverslips (20 x 8 mm), in Leighton tubes containing minimum essential medium with 10% fetal calf serum (MEM-10%), at 37ºC under 5% CO2 atmosphere. After washing three times with PBS, the medium was replaced with 1 ml C. jejuni suspension (6x108 colony forming units) in MEM with 2% fetal calf serum. Cells were incubated for 3 hr at 37ºC and 5% CO2 (infection period), washed 10 times in PBS and reincubated for 4 hr with 1 ml MEM-10% (multiplication period). Then, coverslips were washed three times with PBS and, without fixing, stained with 0.01% acridine orange in Gey's solution for 45 sec, rinsed with Hanks balanced salt solution and counterstained with 0.05% crystal violet in 0.15 N NaCl for 45 sec, mounted on slides and sealed with colorless nail polish and examined under epi-fluorescence microscopy at 400X magnification for screening and 1000X magnification for quantitative evaluation. The invasion rate (no. of invaded cells/total cells examined x 100) was determined by counting at least 200 cells. The average number (± SD) of invading bacteria was estimated in a minimum of 35 invaded HEp-2 cells. All tests were carried out twice in duplicate.

Five invasive and two non-invasive strains were randomly selected for TEM as control tests. Invasion tests were done as described above but using cell monolayers in 35 mm tissue culture dishes. After the multiplication period, the cell monolayers were washed three times with PBS and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 hr, gently scraped and washed with the same buffer under low speed centrifugation. Cell pellets were placed in agar and small blocks were cut and prepared for TEM as described previously for Escherichia coli by U Fagundes-Neto et al. (1995 Acta Paediatrica 84: 453-455).

Results are reported in Table. Sixteen of the 20 C. jejuni strains studied were able to invade HEp-2 cells. In each case, using the AO-CV stain, it was possible to observe intra-cytoplasmatic curved bright green-fluorescing bacteria into the HEp-2 cells. The invasion rate ranged from 8% to 42% whereas the number of bacteria per invaded HEp-2 cell varied from 4.9 ± 2.1 to 38.2 ± 19.2. Fig. 1a shows typical forms of Campylobacter inside the cytoplasm of a Hep-2 cell.

Different techniques have been used to study C. jejuni invasiveness and since the obtained results are not uniform, they do not allow effective or valid comparisons. In our study, invasion by C. jejuni was assessed by a double-stain technique (AO-CV stain) that allows the optical observation (fluorescence microscopy) of internalized bacteria exclusively. This is an advantage of this method since it is difficult to diferentiate extra and intracellular bacteria by employing other methods. The rate of invasion and the number of bacteria per invaded cell observed with this technique are in agreement with a previous report in which the same infection and multiplication periods were used to investigate invasion by May Grünwald-Giemsa staining (Fernández & Trabulsi loc. cit.). G Bukholm and G Kapperud (1987 Infect Immun 55: 2816-2821), using a combination of Nomarski differential interference contrast microscopy and UV incident-light microscopy to discriminate between extra and intracellular bacteria, showed that Campylobacter strains, having Salmonella typhimurium as a coinfectant, could invade cell cultures similarly to that observed with the AO-CV stain technique employed in this study.

The ability to invade, demonstrated by five strains observed with acridine orange-cristal violet staining method, was confirmed by TEM. In all TEM control tests carried out with the invasive strains, but not with the non-invasive ones, it was possible to observe intracellular curved bacteria that seemed to be enclosed into a vacuole, as shown in the Fig. 1b. Similar observations were previously reported by de Mello et al. (loc. cit.).

These results suggest that the acridine orange-crystal violet staining is a suitable technique to study in vitro the ability of C. jejuni to invade cell cultures, similarly to other bacteria as it was previously demonstrated for Arcobacter cryaerophylus by H Fernández et al. (1995 Mem Inst Oswaldo Cruz 90: 633-634).

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