Spirochaete-Like Swimming Mode of Campylobacter Jejuni in a Viscous Environment

J. Med. Microbiol. - Vol. 47 (1998), 521-526 IC 1998 The Pathological Society of Great Britain and Ireland

BACTERIAL PATHOG EN IC ITY

Spirochaete-like swimming mode of Campylobacter jejuni in a viscous environment

MlKA SHIGEMATSU, AKIKO UMEDA, SHUJI FUJIMOTO and KAZUNOBU AMAKO

Department of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka 812-82, Japan

The swimming patterns of Campylobacter jejuni in environments of low and high viscosity were examined by a video tracking method. In media of low viscosity, C. jejuni swam with an average velocity of 39.3pmls with frequent changes in direction. The velocity of C. jejuni increased in a medium at a little higher viscosity than that of a low viscosity buffer. In addition to this, C. jejuni showed a second increase of velocity in media of a high viscosity of about 40 centipoise. The swimming patterns at these two velocity peaks were compared. In the second peak the wild-type C. jejuni exhibited repeated back and forth swimming patterns which were more like the swimming pattern of spirochaetes than that of monotrichous bacteria. Thus C. jejuni may presumably use a different swimming mode in media of high viscosity than the original swimming mode mediated by the propelling force of the flagella. The spiral shape of this bacterium like that of spirochaetes may strongly influence its swimming ability in media of high viscosity such as the mucous layer of the intestinal tract.

Introduction The flagellum of C. jejuni consists of three morphological parts, the helical filament, the hook, which is Campylobacter jejuni, a gram-negative spiral-shaped an intermediate structure connecting the filament and bacterium, is now recognised as the leading cause of the basal structure, and the basal body which is diarrhoea1 disease in man and is often found in the attached to one end of the spiral-shaped bacterial cell intestinal tracts of mammals or birds that are believed [9]. The specialised structure for driving the flagellar to be the epidemiological reservoirs [l, 21. Yet the filament is located in the basal body. Intracellularly, a pathophysiological mechanisms of this infection are complicated structure that may play a role in the still not clearly understood [3]. transfer of information or energy for the movement of the flagellar organelle is found at one end of the cell It has been well documented that, for the colonisation ~91. of the mammalian intestinal tract by this bacterium, the motility mediated by rotation of their flagella is of Various analyses of the bacterial motility of Escher- primary importance [4-71. Morooka et al. reported ichia coli or Salmonella enterica serotype Typhimur- that the non-motile mutant strains they isolated did not ium have revealed that the bacterial cell often changes colonise the intestinal tract of suckling mice when its swimming direction randomly after a brief interval inoculated orally [4, 71. Ueki et al. showed that anti- of vigorous erratic movement which is called flagellar antibodies, especially of the IgM and IgA ‘tumbling’ motion [ 10- 121. The combination of these types, inhibited colonisation [6]. Furthermore, Takata two patterns of movement, swimming and tumbling et al. isolated non-chemotactic mutants of C. jejuni, may lead the bacteria toward suitable substances demonstrated that they did not colonise the mouse called chemo-attractants. The non-chemotactic mutant intestinal tract and excluded the possibility that the strain of C. jejuni is reported to have lost its tumbling flagellar filament is an adhesive organ [5, 81. These motion during swimming [8]. and other reports together indicate that the chemotactic motility by flagella is the major colonisation factor For colonisation of the intestinal epithelium, the of this species. bacteria have to move and adhere in the viscous environment of the thick mucus layer lining the epithelial cell surface of the intestinal tract of humans Received 17 April 1997; revised version accepted 20 Oct. or mammals [ 13- 171. Thus, examining the behaviour 1997. of the bacteria in such a viscous environment is Corresponding author: Dr M. Shigematsu. important for understanding the mechanisms of 522 M. SHIGEMATSU ET AL. colonisation and infection by motile bacteria. To The image of the bacterial movement was tracked by simulate the bacterial motility in the mucous layer, an image analysis system DVS- 1000 (Hamamatsu artificial viscous solutions have been prepared by Photonics, Sizuoka, Japan) on video tape through a dissolving viscous agents in buffer, and motility of the high resolution video camera (monochrome type, bacteria in these viscous environments has then been Hamamatsu Photonics) as described previously [24]. examined. Analysis of the bacterial motility in viscous environments has indicated that the bacteria can move Preparation of viscous solutions well in conditions of high viscosity. Furthermore, it has been recognised as a general behavioural phenom- Viscous solutions were prepared by dissolving an enon of flagellate bacteria that an increase in the appropriate quantity of the following ingredients either swimming velocity is observed as the viscosity in PBS or in modified RF buffer [ 181 comprising 0.1 M increases [ 18-21]. potassium phosphate buffer, pH 7.0, and Tween 80 (Wako Pure Chemical Industries, Osaka, Japan) 2% v/v: The aim of this study was to examine the motility of methylcellulose (MC; Sigma, mol. wt 4000), polyvinyl- C. jejuni in solutions of various viscosities to assess pyrolidone (PVP; Sigma, mol. wt 360 000) and carb- the ecological significance of this organism’s ability to oxymethylcellulose (CMC; Katayama Kagaku Kogyo, remain motile in viscous environments. Osaka, Japan). As these three ingredients all showed similar effects in respect of both the swimming pattern and the speed of C. jejuni, MC was mainly employed in Materials and methods this study. Bacterial strains and culture conditions The kinematic viscosity of the solutions was measured The strain of C. jejuni used in these experiments was with a Cannon-Fenske routine type viscometer (Sibata strain FUM 158432; its origin and characteristics have Chemical App. Mfg Co. Ltd, Japan) and an Ubbelohde been described previously [4, 81. C. jejuni was cultured type viscometer (Kusano Scientific Instrument Mfg Co. at 42°C on Brucella Agar (Difco) supplemented with Ltd, Tokyo, Japan). The density of the solutions was defibrinated sheep blood (Nippon Bio-Supply Center, measured with a Gay-Lussac type flask. The kinematic Tokyo, Japan) 10% or in Brucella Broth (Difco) under viscosity of the sample (in centistokes, cSt) was micro-aerophilic conditions in a GasPak jar without obtained by multiplying the efflux time (in seconds) catalysts (BBL Microbiology Systems, Cockeysville, by the viscometer constant at 30°C. To determine the MD, USA). Vibrio cholerae 0139 strain VO18 [22], viscosity (in centipoise, cP), the kinetic viscosity was Pseudomonas aeruginosa strain K [23] and S. Typhi- multiplied by the density of the solutions (in g/ml). murium strain LT-2 (laboratory collection) were also used in this experiment. These strains were maintained Scanning electron microscopy and cultured on Luria nutrient agar or in broth at 37°C. All these strains originated from our own stock cultures. The shape of the bacteria was observed by scanning electron microscopy (SEM) as described previously [25]. Briefly, the bacteria cultured on a thin agar layer Examination and imaging of the swimming patterns on a glass fibre filter (Whatman GF/F) were fixed and dehydrated in a small petri dish, dried by the critical The bacteria were cultured in liquid media with point drying method, cemented to an aluminium stub constant shaking to their log phase (for 17 h under with silver paste or paper bond and then coated with a micro-aerobic conditions at 42°C for C. jejuni and for thin layer of gold-palladium (Au 6O%, Pd 40%). The 2 h with aeration at 37°C for the other bacteria). The observations were done with an Hitachi HFS-2 field- culture was diluted with 0.1 M phosphate-buffered emission-source SEM operated at 25 kV saline (PBS: NaCl 8 g, NazHP04 29 g, KCl 2 g, KH2PO4 2 g/L; pH 7.0) up to a concentration of lo6 cfu/ml. The method for specimen preparation for Results motility examination has been described previously Swimming pattern and velocity of C. jejuni in a [24]. Briefly, one drop of the bacterial suspension was medium of low viscosity placed on a clean glass slide and then immediately covered with a thin cover glass. Then the edges were In this experiment 0.1 M PBS (pH 7.0) was employed sealed with nail polish to prevent the evaporation of the as a medium of low visosity. The swimming pattern of medium. The movement of the bacteria was examined C. jejuni in this medium was recorded and analysed by with a light microscope (Diaplan Leiz, Ernst Leiz, the video tracking method. The average velocity of the Wetzlar, Germany) under dark field illumination with a wild-type strain was 39.3 pm/s and was slower than 1OX ocular and a 20X objective lens. The observations that of Y cholerae, but faster than that of I? aeruginosa were usually completed within 5 min and it was [24] and S. Typhimurium (22 pm/s). possible to maintain the temperature of the sample on the microscope stage at 30°C. The typical swimming pattern of strain FUM158432 is SPIROCHAETE-LIKE MOVEMENT OF C. JEJUNI 523

Fig. 1. Video tracking image of C. jejuni wild type in PBS. The swimming path was recorded for 15 s. Bar = 100 pm. shown in Fig. 1. Zigzag movements which represent a frequent changes of swimming direction were evident 70 (Fig. 1). The change in direction in the swimming pattern was supposed to be a phenomenon resembling 60 the tumbling motion reported among peritrichously flagellate bacteria [ 10- 121. 50 40 Movement in media of high viscosity 30 As an in-vitro model system of the intestinal mucous layer, the high viscosity media were prepared by 20 dissolving methylcellulose in RF buffer [lS, 19, 261. The experiments were repeated in triplicate and the 10 h swimming patterns of >30 cells were examined in each --.v) experiment.

The average swimming velocities of several flagellate bacteria in the viscous media were plotted against the change in viscosity (Fig. 2). The graphs revealed that as the concentration of methylcellulose increased, the 50 swimming velocity of the bacteria gradually decreased. However, the highest swimming velocity was recorded 40 at a slightly higher viscosity than 1.0 CP (1.5-3 cP) in all the bacterial species examined in this experiment. 30 The graphs of I? cholerae, P aeruginosa and S. 20 Typhimurium had only one peak of highest velocity in the viscous media and this type of graph was 10 designated as the ‘single peak pattern’. On the other hand, in contrast to the other bacteria, C. jejuni showed 0 a second increase in velocity at a higher viscosity of c. 1 10 100 1000 40cP and thus formed a second peak. Thus it was Viscosity (cP) confirmed that C. jejuni had a ‘double peak pattern’ of Fig. 2. The relationship between swimming velocity and swimming velocity as has been reported in campylo- the viscosity of the medium. (a) C. jejuni FUM158432; bacters by other researchers with a different measuring (b) A, I? cholerae, a, I? aeruginosa and 0, S. system [18, 211. Typhimurium. 524 M. SHIGEMATSU ETAL. In these two peaks, as demonstrated in Fig. 3A and Discussion B, the C. jejuni wild-type strain showed different swimming patterns. In the solution of low viscosity, The swimming patterns and the velocity of C. jejuni in at the first peak, the bacteria lost the typical media of low and high viscosity were examined by swimming pattern of the wild type, and swam in a means of a video tracking method. This new method rather straight manner in one direction as was seen in can demonstrate the swimming patterns of a single cell the tracks of non-chemotactic mutants [8]. This of C. jejuni in clear photocopies. swimming pattern was characteristic of the strain up to a viscosity of S40 cP. In the second peak at 40 cP, The analysis of the pattern recorded in a low viscosity the bacteria showed a different pattern from that in salt solution revealed that C. jejuni exhibited frequent the medium of lower viscosity. In this high viscosity directional changes in its swimming pattern. In medium, C. jejuni frequently reversed its swimming contrast to peritrichous bacteria, polar flagellate direction in a back and forth pattern with a relatively monotrichous bacteria are supposed only to change short straight moving path (Fig. 3). In a viscous the rotational direction of their flagella and then move solution >40 cP, the bacteria almost ceased their in another direction. They do not need time to rewind movement. These results revealed that this bacterium the bundles of the flagella in a new direction. In the uses a different moving mode in solutions of high track of the wild-type strain the pattern of the backup viscosity from that generally used in media of low movement and the pause in swimming was rarely seen viscosity. and this might also make it difficult to observe the

Fig. 3. Video tracking images of C. jejuni wild type in highly viscous media. (A) Image of the wild type in the first peak; (B) wild type in the second peak; all were recorded for 3 s. Bars = 100 pm. SPIROCHAETE-LIKE MOVEMENT OF C. JEJUNI 525

Fig. 4. Scanning electron micrograph showing the characteristic spiral shaped C. jejuni with the flagella at both ends of its body. Bar = 500 nm.

tumbling movement with a method other than the bacterium, while at a high viscosity it swam by a video tracking method. different mechanism. Therefore, the assumption was made that the shift in the swimming pattern from a The initial increase in the velocity of the bacteria with flagella-dominated-type to the spiral shape-dominated- the increase in viscosity was confirmed in this study type might occur at a viscosity of c. 40 cP. The video [18-201. This is supposed to be a general behavioural tracking images recorded in these two zones, in the phenomenon of motile bacteria in viscous solutions. first peak and the second peak, showed different However, Lowe et al. have reported that in a viscous patterns. The pattern at the first peak was straight solution prepared with an agent such as ficoll, a high swimming with low tumbling, but at the second peak molecular material without long unbranched chains, the organism showed frequent back and forward Escherichia coli or Streptococcus spp. showed move- movements. This different pattern may represent the ment without any increase in the swimming speed at use of different swimming modes by C. jejuni. high viscosity range [27]. This observation suggested that the increase of swimming velocity in highly This study was supported by a Grant-in-Aid for Scientific Research (B) (06454207) from the Ministry of Education, Science, Culture and viscous solutions is greatly affected by the shape of Sports, Japan. We thank Dr Y. Meno, Faculty of Health and Welfare, the molecule in the solution. This study confirmed Seinan-Jogakuin University for technical advice and Dr B. Quinn for that C. jejuni showed a small increase in velocity at c. critically reading the manuscript. 3.5 CP in ficoll solution (unpublished observations). In addition to this, the velocity of C. jejuni increased again in a high viscosity zone whereas that of the References other bacteria decreased. Ferrero and Lee also demonstrated an increase in the swimming velocity of C. 1. Blaser MJ, LaForce FM, Wilson NA, Wang WL. Reservoirs for human campylobacteriosis. J Infect Dis 1980; 141: jejuni in a medium of high viscosity with a video 665-669. recording method [18]. The increase in the velocity 2. Tauxe RV: Epidemiology of Campylobacter jejuni infections in again at 40 CP is a characteristic phenomenon found in the United States and other industrialized nations. In: Nachamkin I, Blaser MJ, Tompkins LS (eds) Campylobacter C. jejuni. It has also been reported that in a medium jejuni - Current status and future trends. Washington, DC, of high viscosity, the bacteria with spiral shapes, like American Society for Microbiology. 1992: 9- 19. spirochaetes, are able to overcome the problem of the 3. Walker RI, Caldwell MB, Lee EC, Guerry P, Trust TJ, Ruiz Palacios GM. Pathophysiology of Campylobacter enteritis. dampening action of viscous media on their flagella Microbiol Rev 1986; 50: 81-94. because of their characteristic shapes [18, 19, 21, 28, 4. Morooka T, Umeda A, Amako K. Motility as an intestinal 291. C. jejuni has a spiral shaped body and two colonization factor for Campylobacter jejuni. J Gen Microbiol 1985; 131: 1973-1980. flagella, one at each end (Fig. 4). At a low viscosity, 5. Newel1 DG, McBride H, Dolby JM. Investigations on the role C. jejuni behaved just like any other flagellate of flagella in the colonization of infant mice with Campylo- 526 M. SHIGEMATSU ETAL.

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