The e ect of oxygen on the growth and cell morphology of Helicobacter pylori

Transcription

1 FEMS Microbiology Letters 168 (1998) 9^15 The e ect of oxygen on the growth and cell morphology of Helicobacter pylori Gianfranco Donelli a, Paola Matarrese a, Carla Fiorentini a, Benedetto Dainelli b, Tea Taraborelli b, Emanuela Di Campli b, Soraya Di Bartolomeo b, Luigina Cellini b * a Laboratorio di Ultrastrutture, Istituto Superiore di Sanitaé, Viale Regina Elena, Rome, Italy b Dipartimento di Scienze Biomediche, Laboratorio di Batteriologia Medica, Facoltaé di Medicina, Universitaé `G. D'Annunzio', Via dei Vestini 31, Chieti, Italy Received 4 May 1998; received in revised form 31 August 1998; accepted 31 August 1998 Abstract The in vitro effect of progressive oxygen decrease on the growth and morphology of Helicobacter pylori was studied. H. pylori ATCC was used for the experiments. The strain inoculated in Brucella broth plus fetal calf serum was incubated under a controlled atmosphere with oxygen concentration from 5 to 0%. CFU ml 31 and bacterial morphology were detected at the time of spreading and at 24 h, 72 h, 7 days and 14 days. A detailed ultrastructural investigation of the bacterial cells, grown in different experimental conditions, was performed by scanning electron microscopy. Oxygen deprivation produced a rapid reduction of CFU ml 31. In particular, a significant reduction of viable bacteria was recorded at 72 h of incubation in the presence of 1% oxygen and anaerobiosis, and 0 CFU ml 31 was found after 7 days of incubation at the above mentioned oxygen concentrations. The coccoid phenotype was already prevalent after 24 h of incubation with a progressive tendency to aggregate in clusters. These clusters were progressively larger, depending on the reduction of oxygen concentration, since the aggregation phenomenon can be the expression of a hypothesized mechanism of protection among bacterial cells. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Helicobacter pylori; Oxygen; Morphology; Viable but not culturable state 1. Introduction Helicobacter pylori is now de ned as the main etiological agent of active gastritis and peptic ulcer [1^4] and the persistence of this germ in the stomach increases the risk of gastric cancer [5,6]. * Corresponding author. Tel.: +39 (871) ; Fax: +39 (871) ; l.cellini@dsb.unich.it While H. pylori infection is the most common gastrointestinal bacterial disease worldwide, knowledge of its mechanism of transmission is still poor [7^12]. H. pylori, as well as Vibrio-like bacteria, shows two di erent morphologic aspects: the typical spiral form and the coccoid form, the latter observed in vivo [13], and induced in vitro under stress conditions [14^17]. The coccoid forms are not culturable in vitro and, for a long time, investigators have dis / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (98)

2 10 G. Donelli et al. / FEMS Microbiology Letters 168 (1998) 9^15 cussed their viability and their role in germ transmissibility and in infection relapse [13,17^23]. This morphology seems to be related to a speci c form of `dormancy' occurring in non-sporulating bacteria [15,16,24]. According to our data [25], coccoid forms represent the morphological expression of a viable but not culturable (VBNC) state, corresponding to a temporary adaptation to an unsuitable environment. A focal point in the study of VBNC H. pylori is represented by the singling out of the di erent factors which can induce these morphological forms. Moreover, clear di erences in density, DNA contents and secreted proteins among coccoid forms [26] are the consequence of various cultural conditions such as nutrient deprivation, antibiotic addition, unfavorable atmosphere. The aim of this study was to evaluate, in vitro, the e ect of oxygen deprivation on the growth of H. pylori and on the morphology of the bacterial cells. 2. Materials and methods 2.1. Bacterial strain and growth conditions H. pylori ATCC was the standard control strain used for this work. Microorganisms, stored at 370³C by the method of Drumm and Shermann [27], were defrosted to room temperature, and rapidly plated in chocolate agar plus 1% of IsoVitaleX (CA) (Becton Dickinson, Cockeysville, MD, USA) at 37³C for 3 days in a microaerophilic atmosphere (90% N 2, 5% O 2, 5% CO 2, CampyPak Jar, Oxoid Ltd, Basingstoke, UK) Determination of conversion rate under di erent oxygen concentrations Bacteria harvested from almost con uent CA plates were inoculated in Brucella broth (BB, Biolife Italiana, Milan, Italy), supplemented with 2% fetal calf serum (FCS, Seromed, Biochrom KG, Leonorstiftung, Berlin, Germany), and incubated overnight at 37³C in a microaerophilic atmosphere with gentle agitation. At the start of the experiment, the broth culture was divided into tubes of 14 ml each. Groups of four tubes were incubated in separate jars under the following condition of gas mixture: (a) 90% N 2, 5% O 2, 5% CO 2 (standard condition), (b) 91% N 2, 4% O 2, 5% CO 2, (c) 92% N 2, 3% O 2, 5% CO 2, (d) 93% N 2, 2% O 2, 5% CO 2, (e) 94% N 2, 1% O 2, 5% CO 2, (f) 95% N 2, 0% O 2, 5% CO 2 (anaerobiosis). As control, experiments with either 10% CO 2, 85% N 2 and 5% O 2 (A) or 10% CO 2, 90% N 2 and 0% O 2 (B) were also performed. Number of viable bacteria (CFU ml 31 ) and bacterial morphology were determined at time 0 and after 24 h, 72 h, 7 days, and 14 days Viable count The number of viable bacteria was determined by plating 100 Wl of 10-fold serial dilutions in BB plus 2% FCS broth cultures, on CA plates in controlled atmosphere (standard condition) at 37³C for 3 days. For tubes incubated in atmospheres tending to anaerobiosis (2^0% of oxygen) and, for each mixture, after 7 days of incubation, the viable count was also performed by plating on CA in a controlled atmosphere at 37³C for 3 days, both 100 Wl of 10- fold concentrated broth culture and the same aliquot of concentrated broth culture after passage in fresh broth (1 ml of broth culture plus 9 ml of BB+2% FCS and incubated overnight in standard microaerophilic environment at 37³C with gentle agitation). This latter procedure was to evaluate the reactivation of bacteria either in coccoid form or aggregated in clusters Bacterial morphology Bacterial features were studied in Gram modi ed stain with a Zeiss standard microscope at magni cation 1000U. The morphologies, indicated as bacillary (B), or coccoid (C alone, or grouped in CL clusters) were counted as follows: for each broth studied, three slides were read and, for each slide, four elds were counted. Slides were read by two `blind' microbiologists Electron microscopic observations Ten milliliters of each broth culture were centri-

3 G. Donelli et al. / FEMS Microbiology Letters 168 (1998) 9^15 11 fuged for 20 min at 8000Ug and were prepared for scanning electron microscopy (SEM) by xing them with 2.5% glutaraldehyde in 0.1 M cacodylate bu er (ph 7.4) at 4³C for 30 min. After several washes in cold cacodylate, samples were resuspended in 40 Wl of the same bu er. The solutions were dropped on 13-mm polylysine-covered coverslips at room temperature. Then, samples were post xed in 1% OsO 4 for 30 min, dehydrated through graded ethanols, critical point dried in CO 2 and gold coated by sputting. The samples were examined with a Cambridge 360 scanning electron microscope Statistical analysis All experiment data are shown as mean þ S.D. of triplicate determinations and the results are representative of two separate experiments. Student's t-test (`Statistic' program for Macintosh) for correlated samples was used. A P value of less than 0.01 was considered signi cant. 3. Results and discussion The growth of H. pylori ATCC cultures at di erent percentages of oxygen is shown in Fig. 1. Similar results were obtained with 5% and 4% oxygen concentration. Only at these oxygen tensions a signi cant increase of bacterial growth, compared to the starting concentration, was recorded (P ). A peak of 5U10 8 CFU ml 31 was observed at 72 h of incubation and a signi cant reduction of CFU ml 31 until values of 10 6 CFU ml 31 was reached after 14 days (P ). The variations of the morphological aspect of the germ were similar in the above considered environmental conditions (Fig. 2) with prevalence of the coccoid morphology after 24 h of incubation. Moreover, after 7 days of incubation, coccoid H. pylori tended to aggregate in clusters (Fig. 2a,b). These clusters were also observed in conditions c and d (Fig. 2c,d) in which constant values of CFU ml 31 were recorded (Fig. 1), with a reduced culturability in 2% oxygen (condition d). More stringent oxygen Fig. 1. Growth of H. pylori in cultures under di erent oxygen concentrations (a, 5% O 2 ; b, 4% O 2 ; c, 3% O 2 ; d, 2% O 2 ; e, 1% O 2 ; f, 0% O 2 ). Values are plotted as mean þ S.D.

4 12 G. Donelli et al. / FEMS Microbiology Letters 168 (1998) 9^15 Fig. 2. Percentage of H. pylori forms detected under di erent oxygen concentrations (a, 5% O 2 ; b, 4% O 2 ; c, 3% O 2 ; d, 2% O 2 ; e, 1% O 2 ; f, 0% O 2 ). Each value represents an average of triplicate determinations and the results are representative of two separate experiments. The standard deviations range from 30.3 to B, bacillary forms; C, coccoid forms; CL, clusters. deprivation (1% and anaerobiosis, conditions e and f) revealed a reduction of 10-fold of CFU ml 31 at 24 h of incubation and after 7 days of incubation the loss of culturability (0 CFU ml 31 ) was signi cant compared to the previous readings (P ). These latter two condition showed morphologi-

5 G. Donelli et al. / FEMS Microbiology Letters 168 (1998) 9^15 13 Fig. 3. Scanning electron micrographs of H. pylori in di erent oxygen conditions and growth times. A: 4^5% oxygen concentration; B: 2^3% oxygen concentration; C: 1% oxygen and anaerobiosis. Top line (Aa, Ba, Ca): 3 days of bacterial growth; middle line (Ab, Bb, Cb): 7 days of bacterial growth; lower line (Ac, Bc, Cc): 14 days of bacterial growth. Magni cation: 2100U. cally (Fig. 2e,f) a large presence of clusters starting from 24 h of incubation. In particular, at more stringent conditions, in which the percentage of oxygen was from 2% to 0%, an unusual morphology of H. pylori at scanning electron microscopy was detected. Long and undivided rods were observed after 7 days with a tendency to become lamentous (data not shown). The general view of the changes observed in the morphology of H. pylori, grown in di erent oxygen conditions, is presented in Fig. 3. Panel A represents H. pylori cells grown at 4^5% oxygen concentration; panel B at 2^3% oxygen concentration; panel C bacteria grown at 1% oxygen and anaerobic condition. The micrographs on the upper (Aa, Ba, Ca), the middle (Ab, Bb, Cb) and the lower lines (Ca, Cb, Cc) show bacteria cultivated for 3, 7 and 14 days, respectively. The most relevant aspect arising from the comparative analysis of the cells grown in di erent environmental conditions is represented by the increase in the number of clusters as a function of the time of bacterial culture. This phenomenon was more evident with the oxygen deprivation. In particular, in 1^0% oxygen, the presence of clusters (Fig. 2e,f) was earlier than in other oxygen conditions. Regarding the size of clusters, a maximum was reached when the incubation time was prolonged to 14 days and for the oxygen concentration of 1^0%.

6 14 G. Donelli et al. / FEMS Microbiology Letters 168 (1998) 9^15 Fig. 4. Growth of H. pylori in cultures detected in standard conditions (A) and in anerobiosis (B). Values are plotted as mean þ S.D. Fig. 4 shows bacterial growth in both anaerobiosis and microaerophilic atmosphere obtained using 85% N 2, 5% O 2 and 10% CO 2. In this microenvironment with a percentage of CO 2 higher (10%) than that used for the above reported experiments (5%), CFU ml 31 decreased more rapidly and no viable bacteria were recorded after 14 days of incubation. In conclusion, our results show that the presence of 5^4% oxygen in the gas mixture reveals a good growth of H. pylori, with an increase of 100-fold of the starting concentration after 72 h of incubation. The e ect of oxygen decrease (3^2%) shows, after each control time, a relatively constant value of CFU ml 31. In fact, at 14 days of incubation bacterial growth was still observed at the above mentioned oxygen concentration. Further oxygen deprivation produced a rapid reduction of CFU ml 31. In particular, a signi cant reduction of viable bacteria (about two steps of concentration) was recorded at 72 h of incubation in the presence of 1% oxygen and anaerobiosis, and 0 CFU ml 31 was found after 7 days of incubation at the above mentioned oxygen concentrations. Five instead of 10% CO 2 enhanced the tolerance to the presence of toxic catabolic products and to the starvation. Particularly interesting are the morphological changes observed at the ultrastructural level. Coccoid forms are more rapidly induced at low oxygen concentration with a tendency to aggregate with each other to form progressively larger clusters in accordance with recent data on adhesive properties of coccoid versus spiral forms of H. pylori [20]. Moreover, the presence of aggregate cocci can be interpreted as the modi cation of the behavioral answers of the germs which, in unfavorable conditions, tends to overcome the toxicity of the medium by their clustering. Krieg and Ho man [28] studied the e ect of a high density of bacterial population on its own self-protection against the toxicity created in the medium because of increased oxygen; the more the broth culture is concentrated, the higher the protection of cells. An analogous mechanism could be effective in our opposite experimental conditions of oxygen deprivation in which the presence of clusters, more evident after 7 days of incubation, can express the tendency of cells to protect each other.

7 G. Donelli et al. / FEMS Microbiology Letters 168 (1998) 9^15 15 Acknowledgments This work was supported by Grant CU CT04 from the Consiglio Nazionale delle Ricerche and of the Ministero dell'universitaé e della Ricerca Scienti ca e Tecnologica (60%). References [1] Goodwin, C.S., Mendall, M.M. and North eld, T.C. (1997) Helicobacter pylori infection. Lancet 349, 265^269. [2] Labigne, A. and de Reuse, H. (1996) Determinants of Helicobacter pylori pathogenicity. Infect. Agents Dis. 5, 191^202. [3] Lee, A., Fox, J.G. and Hazell, S. (1993) Pathogenicity of Helicobacter pylori: a perspective. Infect. Immun. 61, 1601^ [4] Wyatt, J.I. and Dixon, M.F. (1988) Chronic gastritis ^ a pathogenetic approach. J. Pathol. 154, 113^124. [5] Asaka, M., Kimura, T., Kato, M., Kudo, M., Miki, K., Ogoshi, K., Kato, T., Tatsuta, M. and Graham, D.J. (1994) Possible role of Helicobacter infection in early gastric cancer development. Cancer 73, 2691^2694. [6] Forman, D. (1996) Helicobacter pylori and gastric cancer. Scand. J. Gastroenterol. 251, 48^51. [7] Hill, M. (1997) The microbiology of Helicobacter pylori. Biomed. Pharmacother. 51, 161^163. [8] Mendall, M.A. (1997) Transmission of Helicobacter pylori. Semin. Gastrointest. Dis. 8, 113^123. [9] Mendall, M.A. and North eld, T.C. (1995) Transmission of Helicobacter pylori infection. Gut 37, 1^3. [10] Namavar, F., Roosendaal, R., Kuipers, E.J., de Groot, P., van der Bije, M.W., Pena, A.S. and de Graa, J. (1995) Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis. Eur. J. Clin. Microbiol. Infect. Dis. 14, 234^237. [11] Sahay, P., West, A.P., Hawkey, P.M. and Axon, A.T.R. (1995) Isolation of Helicobacter pylori from faeces. J. Infect. 30, 262^263. [12] Sipponen, P. (1997) Helicobacter pylori gastritis ^ epidemiology. J. Gastroenterol. 32, 273^277. [13] Chan, W.J., Hui, P.K., Leung, K.M., Chow, J., Kwok, F. and Ng, C.S. (1994) Coccoid forms of Helicobacter pylori in the human stomach. Am. J. Clin. Pathol. 102, 503^507. [14] Bena ëssa, M., Babin, P., Quellard, N., Pezennec, L., Cenatiempo, Y. and Faucheére, J.L. (1996) Changes in Helicobacter pylori ultrastructure and antigens during conversion from the bacillary to the coccoid form. Infect. Immun. 64, 2331^2335. [15] Cellini, L. (1996) Coccoid forms of Helicobacter pylori. J. Infect. Dis. 173, [16] Cellini, L., Allocati, N., Di Campli, E. and Dainelli, B. (1994) Helicobacter pylori: a ckle germ. Microbiol. Immunol. 38, 25^30. [17] Kusters, J.G., Gerrits, M.M., Van Strijp, J.A.G. and Vandenbroucke-Grauls, C.M.J.E. (1997) Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect. Immun. 65, 3672^3679. [18] Berry, V., Jennings, K. and Woodnutt, G. (1995) Bactericidal and morphological e ects of amoxicillin on Helicobacter pylori. Antimicrob. Agents Chemother. 39, 1859^1861. [19] Bode, G., Mauch, F. and Malfertheiner, P. (1993) The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol. Infect. 111, 483^490. [20] Cole, S.P., Cirillo, D., Kagno, M.F., Guiney, D.G. and Eckmann, L. (1997) Coccoid and spiral Helicobacter pylori di er in their abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion. Infect. Immun. 65, 843^846. [21] Hua, J. and Ho, B. (1996) Is the coccoid form of Helicobacter pylori viable? Microbios 87, 103^112. [22] Narikawa, S., Kawai, S., Aoshima, H., Kawamata, O., Kawaguchi, R., Hikiji, K., Kato, M., Iino, S. and Mizushima, Y. (1997) Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori. Clin. Diagn. Lab. Immunol. 4, 285^290. [23] Soërberg, M., Nilsson, M., Hanberger, H. and Nilsson, L.E. (1996) Morphologic conversion of Helicobacter pylori from bacillary to coccoid form. Eur. J. Clin. Microbiol. Infect. Dis. 15, 216^219. [24] Cellini, L., Allocati, N., Angelucci, D., Iezzi, T., Di Campli, E., Marzio, L. and Dainelli, B. (1994) Coccoid Helicobacter pylori not culturable in vitro reverts in mice. Microbiol. Immunol. 38, 843^850. [25] Cellini, L., Robu o, I., Di Campli, E., Di Bartolomeo, S., Taraborelli, T. and Dainelli, B. (1998) Recovery of Helicobacter pylori ATCC43504 from a viable but not culturable state: regrowth or resuscitation? APMIS 106, 571^579. [26] Enroth, H., Wreiber, K. and Engstrand, L. (1996) Comparisons of density, DNA content and secreted proteins in wellde ned coccoid forms of Helicobacter pylori. Gut 39 (Suppl. 2), A55^A56. [27] Drumm, B. and Sherman, P. (1989) Long-term storage of Campylobacter pylori. J. Clin. Microbiol. 27, 1655^1656. [28] Krieg, N.R. and Ho man, P.S. (1986) Microaerophilic and oxygen toxicity. Annu. Rev. Microbiol. 40, 107^130.