Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2003
Volume 6
Issue 2
Topic:
Biology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Kot B. , Wolska K. 2003. ADHESION OF YERSINIA ENTEROCOLITICA TO CHEEK MUCOUS MEMBRANE CELLS OBTAINED FROM HUMANS AND PIGS, EJPAU 6(2), #01.
Available Online: http://www.ejpau.media.pl/volume6/issue2/biology/art-01.html

ADHESION OF YERSINIA ENTEROCOLITICA TO CHEEK MUCOUS MEMBRANE CELLS OBTAINED FROM HUMANS AND PIGS

Barbara Kot, Katarzyna Wolska

 

ABSTRACT

Evaluation of adherence of Y. enterocolitica strains isolated from humans and pigs to cheek epithelium cells obtained from humans and pigs was the aim of present study. 51 strains of Yersinia enterocolitica including 34 isolated from the faeces of people who showed typical symptoms of intestinal yersiniosis and 17 isolated from pigs were used in the study. The all Y. enterocolitica strains which were used in the investigation showed the ability of adherence to the cells of cheek epithelium from humans. The cells of Y. enterocolitica strains which had yadA gene more numerously adhered to the cells of cheek epithelium from humans than the cells of strains which did not have yadA gene.

Key words: Yersinia enterocolitica, adhesion.

INTRODUCTION

The interaction between bacteria or bacterial products and host tissues or soluble proteins is crucial during infectious diseases, both for primary adhesion and invasion of the microorganism into the host and for tissue – specific colonization and disease tropism.

A variety of bacteria have been shown to bind host proteins, in particular, the extracellular matrix (EMC) proteins collagen [4, 23], laminin [26], and fibronectin [29].

Yersinia enterocolitica is a common pathogen for both humans and animals. Y. enterocolitica is ubiquitous and is most frequently isolated from the environment, e.g., water, soil, and plant surface [16], from food [6], or from pigs, which are an important reservoir for food-borne infections of humans with pathogenic Y. enterocolitica 0:3 and 0:9 strains [11]. Enteropathogenic Yersinia species can cause different types of diseases; ranging from mild diarrhoea to septicemia; mesenteric lymphadenitis and related diseases that mimic the symptoms of appendicitis are common [2]. The intestinal infection caused by these microorganisms can cause disease sequelae such as reactive arthritis, erythema nodosum, and uveitis [2]. Binding of Y. enterocolitica to epithelial cells, to several types of collagen, to fibronectin, and to laminin is YadA - mediated [4, 23, 28]. In addition to having YadA-mediated binding properties, Y. enterocolitica possesses other molec ules, such as invasin, Ail, and type 3 fimbriae, which mediate binding to a number of targets [17, 27].

Evaluation of adherence of Y. enterocolitica strains isolated from humans and pigs to cheek epithelium cells obtained from humans and pigs was the aim of present study.

MATERIALS AND METHODS

Bacterial strains

51 strains of Yersinia enterocolitica including 34 isolated from the faeces of people who showed typical symptoms of intestinal yersiniosis and 17 isolated from pigs were used in the study. These strains were previously described [13, 14, 15]. The presence of yadA gene in tested strains was determined previously by using PCR method [14, 15].

The cells of cheek epithelium

The cells of cheek epithelium were obtained from 6 healthy humans. The cells of cheek epithelium from pigs were received directly after exanguination of pigs.

The cells of cheek epithelium were suspended in sterile phosphate buffered saline (PBS), pH 7.2 and sedimented by centrifugation (2500 g, 10 min). The cells of cheek epithelium were washed three times with sterile PBS and finally suspened in PBS. The suspension of epithelium cells about 105 per ml was used in adherence test.

Adhesion of Y. enterocolitica to the cells of cheek epithelium

The tested Y. enterocolitica strains were incubated overnight on TSA agar at 25°C. After that the bacterial cells were suspened in PBS pH 7.2. The bacterial density was adjusted turbidimetrically to approximately 109 bacteria per ml (A600 0.625) and appropriate concentrations (108 per ml) were subsequently obtained by 10-fold dilution in sterile buffer.

1 ml of suspension of bacterial cells (108 per ml) was added to 1 ml of suspension of epithelium cells (105 per ml) and incubated with shaking for 2 h in 37°C waterbath. The control test was made by incubation of cheek epithelium cells without Y. enterocolitica cells in the same conditions.

After incubation, in the aim to remove not adhered bacterial cells the mixture of the epithelium cells and bacteria cells was centrifugated (2500 g, 10 min) and the sediment was washed three times with sterile PBS. 30µl of the sediment was transferred on slide, smeared and then Gram stained. Adherent bacterial cells were counted in relation to 50 of the cheek epithelium cells. The results were expressed as mean number of adherent bacteria to one epithelium cell. Statistical calculation was performed by using Levene test.

RESULTS

Table 1 shows adherence of Y. enterocolitica isolated from humans and pigs to the human cells of cheek epithelium taking into consideration of presence or absence yadA gene. The interval >100-150 which determined number of bacterial cells adherent to one epithelium cell was represented by the most number of Y. enterocolitica strains both isolated from humans and pigs (Table 1 and Figure 1). The numbers >150-200 and >200 of the bacterial cells adhering to one epithelium cells was also characteristic of some Y. enterocolitica strains obtained from humans and one strain from pigs. It was found that these strains had yad A gene (Table 1). Whereas the strains of Y. enterocolitica with no yadA gene present represented the interval >0-50 and >50-100. Two strains of Y. enterocolitica which differ in presence of yadA gene were used for further i nvestigation. Adhesion of bacterial cells to the human cells of cheek epithelium obtained from four donors and to the cells of cheek epithelium from four pigs was evaluated. The tested Y. enterocolitica strains differed in number of adhering cells. The mean values of the cells of Y. enterocolitica 97 strain (yadA+) adherence were 158 in the case of the cheek epithelium from humans and 127.82 in the case of the cheek epithelium from pigs. The mean values of adherence of Y. enterocolitica 97 cells (yadA+) both to cheek epithelium cells from humans and from pigs were significantly higher than the mean values of adherence of Y. enterocolitica 8 cells (yadA-) which were 89.35 in the case of the cheek epithelium from humans and 50.85 in the case of the cheek epithelium from pigs. Adhesion of Y. enterocolitica 8 strain (yadA-) to the human cells of cheek epithelium was significantly higher than adhesion to the cells of cheek epithelium from pigs at p£ 0.016.

Table 1. Adhesion of Y. enterocolitica to the cells of the cheek epithelium from humans taking into consideration presence of yadA gene and origin of strains

Mean number of adherent bacteria cells to cheek epithelium cells

Number of Y. enterocolitica strains adherent to cheek epithelium
of human cells

Strains isolated from humans

Strains isolated from pigs

YadA+

YadA-

Total

YadA+

YadA-

Total

>0 - 50

0

1

1

0

1

1

>50 -100

0

10

10

0

6

6

>100 - 150

11

3

14

8

1

9

>150 - 200

7

0

7

1

0

1

>200

2

0

2

0

0

0

Fig. 1. Adhesion of Y. enterocolitica to the cells of cheek epithelium from humans taking into consideration origin of strains

DISCUSSION

The ability to adhere to mucosal surfaces is regarded as an important prerequisite for microorganisms which cause disease by invasion of the intestinal mucosa. Enteropathogenic strains of Y. enterocolitica are able to adhere to cultured epithelial cells. In contrast, non-pathogenic strains of the same species do not adhere [5]. Cellular penetration of enteropathogenic Yersinia species is mediated mainly by invasin, the product of a chromosomal inv gene [21] that initiates entry by binding to mammalian cell receptors belonging to the integrin family [8]. Y. enterocolitica also has the chromosomal ail gene product, which facilitates adhesion and invasion of bacteria [17]. Invasin is expressed early in infection and is probably important for initiation of gastrointestinal infection, Ail is induced later and is important for extracellular spread [7]. In addition to chromosomally encoded factors, the Yersinia virulence plasmid (pYV)-encoded factors also play a role in the bacterium's adhesive properties; pYV–positive Y. enterocolitica strains adhere better to intestinal tissue in vitro than the corresponding isogenic pYV–negative strains [19]. The pYV plasmid encodes a number of Yersinia outer membrane proteins (Yops) which are important virulence determinants. One of these proteins, YadA (Yersinia adhesin), is largely responsible for the adhesion of Y. enterocolitica to intestinal tissue in vitro [20]. Furthermore, YadA has been associated with numerous phenomena: autoagglutination of Y. enterocolitica and of Y. pseudotuberculosis [25], serum resistance of Y. enterocolitica [22], expression of fibrils on the bacterial surface [12] and binding to collagens [4, 23].

The all Y. enterocolitica strains which were used in the investigation showed the ability of adherence to the cells of cheek epithelium from humans. The all investigated Y. enterocolitica strains had chromosomal ail gene, which was determined previously [30]. The cells of Y. enterocolitica strains which had yadA gene adhered more frequently to the cells of cheek epithelium from humans than the cells of strains which did not have yadA gene. The mean value of the cells of Y. enterocolitica 97 strain adherence, which had yadA gene was statisticaly significantly higher than the mean value of adherence of the cells of Y. enterocolitica 8 strain, which did not have yadA gene both in case of the cheek epithelium from humans and from pigs. In case of the Y. enterocolitica 97 strain significant difference between adherence of the bacteria cells of this strain to the cheek epithelium from humans and pigs was not obtained. W hereas the cells of Y. enterocolitica 8 strain which did not have yadA gene numerously adhered to the human cells of cheek epithelium. The investigation of other authors concerning the occurrence of Y. enterocolitica in slaughter pigs demonstrated that the largest percentage of Y. enterocolitica isolation obtained from tongues swabs, following from throat and tonsils. Among isolated strains dominated the strains belonging to 0:3 serotype, biotype 4 [9].

Mouth cavity of slaughter pigs is a main source of Y. enterocolitica strains considered as pathogenic for humans [9]. Nesbakken and Kapperud [18] investigated slaughter pigs in Norway, obtained 83.3% of positive samples from mouth cavity of pigs. Andersen [1] obtained this microorganism in 25% tested samples in throat swabs. Quantitative investigations performed by Shiozawa et al. [24] showed that larger numbers of Y. enterocolitica cells were present in tonsils than in faeces of these same pigs. Jakubczak et al. [10] indicated the presence of Y. enterocolitica among other things in tonsils of the pigs which were experimentally intravenously and by a stomach infected but he did not indicate Y. enterocolitica in gastric contents, duodenum, jejunum, colon or mesenteric lymph nodes. Investigations of Doyle et al. [3] and Kapperud [11] indicated that mouth cavity of slaughter pigs is the main source of carcass contamination during exenteration.

CONCLUSIONS

  1. The investigated Y. enterocolitica strains showed the ability of adherence to the cells of cheek epithelium from humans.

  2. The cells of Y. enterocolitica strains having yadA gene more numerously adhered to the cells of cheek epithelium from humans than the cells of Y. enterocolitica strains without yadA gene.

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Barbara Kot, Katarzyna Wolska
Department of Microbiology
University of Podlasie
ul. B. Prusa 12, 08-110 Siedlce, Poland
e-mail: bkot@ap.siedlce.pl

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