Competitive exclusion of Campylobacter jejuni by kefir fermented milk

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1 Annals of Microbiology, 53, (2003) Competitive exclusion of Campylobacter jejuni by kefir fermented milk C. ZACCONI *, G. SCOLARI, M. VESCOVO, P.G. SARRA Istituto di Microbiologia, Facoltà d Agraria, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, Piacenza, Italy Abstract - Competitive exclusion of Campylobacter jejuni by kefir fermented milk was checked in experimental chicks. Fresh and frozen kefir fermented milk were employed as competitive treatments. Campylobacter jejuni ATCC strain was used as infective treatment. Data from the various trials allowed to determine Infection Factor (IF) and Protection Factor (PF) values and to evidentiate that kefir administration is an effective treatment to prevent Campylobacter jejuni colonization in chick caecum. Key words: kefir fermented milk, Campylobacter jejuni, competitive exclusion, chicks. INTRODUCTION Campylobacter jejuni is recognized as the principal cause of human acute bacterial gastroenteritis (Skirrow, 1991; Skirrow and Blaser, 1992) at the infectious dose of 500 CFU/person (Park, 2002). It was demonstrated that this organism occurs at high percentage (30 to 100%) in poultry (Shane, 1992; Humphrey et al., 1993; Glünder, 1994; Doboszynska and Drzewiecka, 1997), which is the primary source of infection (Harris et al., 1986). The human infections due to Campylobacter spp. are more numerous compared to those due to Salmonella and Shigella in many countries (Stern and Kazmi, 1989). During 1996, Campylobacter spp. caused 46% of bacterial gastroenteritis cases in United States, whereas Salmonella (28%) and Shigella (17%) were responsible for significantly fewer (Alketruse et al., 1999). Several vehicles of illness comprise raw, barbecued or undercooked chickens or eating-foods which, during storage or preparation, have been cross-contamined by raw chickens (Beutling, 1998). Various attempts have been made to contrast the presence of such a pathogen in poultry, including the use of hurdles to exclude risk factors (Person and Healing, *Corresponding author: Phone: ; Fax: ; carla.zacconi@unicatt.it 179

2 1992; Ruairi-Bruga and Regan, 1996). Nurmi and Rantala (1973) were the first authors introducing the concept of competitive exclusion to reduce Salmonella infection rate. Further studies were carried out to verify the effectiveness of competitive exclusion against Salmonella and Campylobacter by the use of either undefined bacterial mixtures from the caecal content of adult chickens (Mulder and Bolder, 1991; Aho et al., 1991) or defined microflora (Schoeni and Doyle, 1992; Schoeni and Wong, 1994; Mead et al., 1996). However, the administration of fecal suspensions could cause health and safety problems to animals with an evident decrease of animal weight, while some associations of lactic microflora could be suitable to express competitive exclusion against intestinal pathogens. Kefir is a natural complex association of microorganisms, not reproducible in vitro (Halle et al., 1994); yeasts, lactic acid bacteria (LAB) and acetic bacteria are generally found as microflora components (Angulo et al., 1993; Bottazzi et al., 1994). The stability of the microbial culture is demonstrated by the constance of the microflora composition although milk fermentation occurs in non aseptic conditions; the probable reason of its stability relies on the intricate complex sinergistic and inhibitory interactions among the species of the kefir microflora. The inhibitory power of kefir was well demonstrated by Garrote et al. (2000) and, particularly, the competitive action of kefir against Salmonella kedougou was described in chicks (Zacconi et al., 1995). The present study aims to verify the competitive exclusion activity of kefir in vivo by assessing the reduction of Campylobacter jejuni colonization of chicks. The conditions of kefir administration to chicks were optimized and the effect of fresh and frozen kefir preparations was compared. MATERIALS AND METHODS Strains and culture conditions. Campylobacter jejuni ATCC strain, resistant to nalidixic acid was used. It was cultured in Tryptone Soya Agar (Oxoid LTD, Basingstoke, Hampshire, England), added of sheep blood (10%) and incubated at 44 C under microaerophilic conditions (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 48 h. The strain was stored in the Institute Collection in liquid nitrogen vapours. Kefir grains were obtained from different private households in Italy. Stock kefir grains were maintained at 80 C and subcultured in pasteurized milk at room temperature. To obtain fermented milks, active kefir grains were washed with sterile distilled water, inoculated into fresh pasteurized milk and incubated at room temperature. The fermented milk was separated from kefir grains through a sterilized sieve and directly employed for some trials or stored at 20 C, for 7 days for the use in other trials. Esperimental animals. Trials were carried out on newly hatched chicks Ross 1207, males, directly obtained from an industrial hatchery. Chicks were maintained in aluminium cages under aseptic conditions at 22 (± 1) C, 50 (± 10) % relative humidity and with 12 h light-dark cycle. The pelletized diet of chicks, free from antimicrobial substances, was sterilized by γ-ray radiation and supplied to chicks ad libitum during all the trials from the second day of life. 180 C. ZACCONI et al.

3 Competitive treatments. In order to compare the effectiveness of fresh and frozen (7 days, -20 C storage) kefir fermented milk, competitive treatments were performed by fermented milks, supplied to chicks as liquid diet. Infective treatment. 0.1 ml of an overnight culture (10 7 CFU/ml) of Campylobacter jejuni ATCC strain was orally administered to chicks (body weight 40 g) as infective treatment, on the first day of life. Experimental conditions. Chicks within individual experiments were randomly divided into three groups (1 control and 2 treated groups) each consisting of 10 animals, bred as above described. Each experiment was performed as three replicates (3 controls and 6 treated groups) for 12 days and was repeated five times, employing the same type of chicks from different hatchings. Sterile water was administered to the control group as unique beverage from the first day of chick life up to the whole experimental period, while the treated groups were administered with fresh and frozen kefir fermented milks after dilution (1:1) in sterile water. Different series of experiments (a, b, c) were carried out according to the scheme: a) the control group was treated only with the infective treatment on the first day of chick life: a treated group was administered either with infective dose or fresh kefir fermented milk (1 ml) on the first day of chick life; a second group was treated with both infective dose and frozen kefir fermented milk after storage at 20 C for 7 days, on the first day of chick life; b) a serie of experiments was performed under the same conditions of the experiment a) except for the administration of protective treatments as liquid diet daily, ad libitum; c) further experiments were conducted without infective treatment administering fresh and frozen kefir fermented milks on the first day of life, in order to verify the efficacy of kefir administration in the control of naturally contaminating pathogenic microflora in breedings. Bacteriological analysis. After 12 days, chicks of each group were sacrified and caeca were aseptically removed. Caecal contents (1 g) were homogenized in sterile peptone water (0.1%) and decimal dilutions prepared for surface plating on Campylobacter agar base (Karmali) added of Campylobacter Selective Supplement (Karmali) (OXOID, Unipath Ltd, Basingstoke, England); selective counts of added Campylobacter jejuni cells were performed by the addition to the culture media of nalidixic acid (40 µg/ml) (Sigma Aldrich, Milano, Italy). Plates were incubated as decrived above. The same medium without nalidixic acid was used to detect the total population of Campylobacter spp. Total coliforms were evaluated on Violet Red Bile Agar (VRBA) (OXOID) after 24 h incubation at 37 C and rod lactic acid bacteria on MRS (DIFCO, Michigan, USA), ph 5.5, after 48 h incubation at 37 C under anaerobiosis. Evaluation of animal weight increase. The weight increase of each animal was evaluated calculating the difference of the body weight on the 12 th day with respect to that of the first day of life. Feed conversion indexes were calculated for each Ann. Microbiol., 53, (2003) 181

4 group of chicks as ratio between weight increase and feed consumption, including kefir for treated groups. Data elaboration. Results obtained from the Campylobacter jejuni counts of either control or treated groups, were elaborated according to the Mead et al. formula (1989) to calculate the Infection Factor (IF) and the Protection Factor (PF) parameters, as proposed by Pivnick et al. (1984). The IF relates to the degree of infection and represents the mean values of the log CFU/g caecal content of Campylobacter jejuni of each animal within the various groups; the higher the number of C. jejuni/g of caecum, the higher the IF. Treatment efficacy is expressed as PF which is the ratio between the IF of the control compared to that of the treated group. High PF values denote high levels of protection; a PF 1 indicates no protection. IF and PF were calculated for each treatment in each trial by using the corresponding controls. The combined results were averaged and presented with standard deviations (SD). PF values of treated groups were analyzed by the student t test. The results obtained by coliforms and LAB counts from control and fresh kefir treated animals, were elaborated by statistical analysis (t test for indipendent samples, SPSS 9.0), testing the null hypothesis of equality of mean values of two groups. Weight values of each animal were statistically elaborated (Anova, SPSS 9.0). The significativity of the differences between chicks groups was tested by Tukey s test. RESULTS AND DISCUSSION The activity of competitive exclusion by undefined microflora of kefir fermented milk against the emergent pathogen Campylobacter jejuni was evaluated in chicks. The antagonistic activity of fresh fermented milk previously demonstrated against Salmonella kedougou in chicks (Zacconi et al., 1995) was related to its microflora; in fact the acidified milk used as competitive treatment did not show any effect. Frozen kefir was here employed to verify if the freezing process could decrease the possible antagonistic activity of kefir microflora; this is an important aspect as the potential application of such a protective culture to chicks during breeding or some days before slaughtering of adult animals, requires the possibility of an easy commercialization. The administration of kefir fermented milk gave satisfactory results in the prevention of Campylobacter spp. colonization, either in chicks treated with the infective agent or in not infected chicks. Levels of Campylobacter jejuni ATCC contamination in infected chicks caeca did not evidentiate; substantial differences between the strong inhibitory effects of both fresh and frozen kefir fermented milks. Campylobacter jejuni counts showed a reduction in kefir treated chicks up to 6-7 log CFU/g caecal content. Correspondingly, the IF and PF values related to the control and treated groups are shown in Table 1. Statistically significative differences were detected between the PF values of chicks fed with fresh or frozen kefir fermented milk when chicks were treated only on the first day of life (experiment a); on the con- 182 C. ZACCONI et al.

5 TABLE 1 Mean values ± SD of IF and PF as inhibition parameters of Campylobacter jejuni ATCC in chicks artificially infected by the pathogen without (control) and with treatment of fresh or frozen kefir fermented milks on the first day of life and daily ad libitum Chick groups IF PF Significativity First day of life Control 7.8 ± 0.5 n.d. Fresh kefir fermented milk 1.2 ± ± 0.4 p < 0.05 Frozen kefir fermented milk 1.3 ± ± 0.5 p < 0.05 Daily ad libitum Control 8.3 ± 0.4 n.d. Fresh kefir fermented milk 1.2 ± ± 0.6 n.s. Frozen kefir fermented milk 1.3 ± ± 0.2 n.s. n.d.: PF cannot be calculated for the control. trary, no significative differences were detectable in chicks treated daily ad libitum (experiment b). Moreover, the different way of kefir administration (experiments a and b) gave significative differences only when frozen kefir was employed. It is noteworthy that trials performed without infective treatment (experiment c) demonstrated the ability of kefir to contrast the growth of Campylobacter spp. normally present in chick caeca; data in Table 2 indicate a difference of 3 log CFU/g caecal content between chicks treated with fresh and frozen kefir fermented milk with respect to the controls. The remarkable competitive exclusion activity of kefir detected in chick groups treated with fresh and frozen kefir, probably relies on the complexity of microflora association in such a fermented milk. Similar results were obtained by Schoeni and Wong (1994), but at higher PF values and lower IF values; this confirms the ability of kefir to inhibit Campylobacter jejuni growth also at high infec- TABLE 2 Effect of fresh and frozen kefir fermented milks on Campylobacter spp. in chicks without infective treatment. Fresh and frozen fermented milks were administered on the first day of life Chick groups Campylobacter spp. (CFU/g caecal content) a First day of life 12 days Control ± ± 0.3 Fresh kefir fermented milk ± ± 2.0 Frozen kefir fermented milk ± ± 1.3 a Mean ± SD. Ann. Microbiol., 53, (2003) 183

6 tious doses (present results). This is in accordance with the results of other authors (Mead et al., 1996) who demonstrated that the complexity of microflora is the responsible for competitive exclusion activity against Salmonella in caeca of poultry. In order to verify the ability of kefir fermented milk to modulate caecal microflora, the effect of kefir treatments of chicks on total coliforms and rod LAB was also checked. In Table 3 are reported the mean values, standard deviation and variation coefficents of rod LAB and total coliforms CFU/g caecal content, related to animals treated with fresh kefir. Mean values and standard deviation were calculated for each group of animals, for both microbial population. The variation coefficient (CV), calculated as ratio between SD and mean values (CV = σ/µ 100), resulted higher in treated groups. According to the t test, the kefir fermented milk modulated significantly only the levels of total coliforms population, while the LAB rods appear to be insensitive to the treatment. However, during an experiment without infective treatment, the totality of control chicks died at the third day of life, while all the treated chicks were alive and healthy up to the end of the trials. From successive microbiological analysis of caeca it was not possible to isolate pathogenic microorganisms responsible for chicks death, but the total absence of LAB was observed, already on the first day of life. On the contrary, treated chicks showed a level of LAB usually detected at the end of the various other experiments. The analysis of variance of chicks weight increase demonstrated a positive effect of fresh and frozen kefir milk on animal growth in infected chicks (Table 4), with an index of feed conversion of 0.51% in treated chicks compared to control group (0.30%). Comparable results were obtained in non infected chicks (data not shown). The lower weight increase observed in infected animals may be due to a trophic competition for nutrients between host intestinal epithelial cells and the added pathogenic microorganisms associated to the digestive tract. However, the difference in weight increase and relative index of feed conversion, observed also in the TABLE 3 Presence of rod lactic acid bacteria and total coliforms in infected chicks (control) and treated with fresh kefir on the first day of life Chick groups (log CFU/g) a Coefficient of Significativity variation Rod lactic acid bacteria Fresh kefir ± n.s. fermented milk Control ± n.s. Total coliforms Fresh kefir ± p < 0.01 fermented milk Control ± p < 0.01 a Mean ± SD. 184 C. ZACCONI et al.

7 TABLE 4 Significativity of average differences in weight increase and relative indexes of feed conversion among the differently treated groups of infected chicks Chick groups Chick Weight increase Significativity a Feed conversion number average (g) ± SD index Control ± 0.68 A 0.30 Treated with fresh ± 0.52 B 0.45 kefir on the first day of life Treated with frozen ± 0.43 C 0.49 kefir on the first day of life Treated with fresh ± 0.98 C 0.51 kefir daily Treated with frozen ± 0.77 B 0.46 kefir daily a Different letters show averages significantly different at 95%, according to Tukey s test. experiments with not infected chicks, suggests a probable contribution of vitamins and enzymes of kefir. In conclusion the above data demonstrated a strong colonization of chicks caeca by Campylobacter jejuni in the infected chicks ( CFU/g of caecal content) and a relevant antagonistic activity of kefir fermented milk against the considered pathogen was observed. The same activity was detectable also against the Campylobacter spp. naturally occurring in chicks. Moreover kefir had a positive effect on chick weight increase and the relative index of feed conversion, but it showed a limited capacity to modulate other microbial groups. Fresh and frozen kefir could have interesting applications in the control of the diffusion of pathogenic microorganisms in poultry breedings; particularly, frozen kefir may be more easily employed. Further investigations are required in order to elucidate the nature of competitive exclusion. Studies are in progress to verify the capacity of kefir microbial components to modulate the intestinal microflora. Previous researches (Zacconi et al., 1999) demonstrated that single cultures of Lactobacillus salivarius A23 administered to chicks at the first day of life, were able to colonize the intestinal tract and to influence the indigenous microflora, but showed a weak competitive exclusion REFERENCES Aho M., Nuotio L., Nurmi E., Kiiskinen T. (1991). Development of a competitive exclusion flora active against campylobacters in poultry. Microb. Ecol. Health Dis., 4: Special S79. Alketruse S.F., Stern N.J., Fields P.I., Swerdlow D.L. (1999) Campyolobacter jejuni an emerging foodborne pathogen. Emerg. Infect. Dis., 5: Angulo L., Lopez E., Lema C. (1993). Microflora present in kefir grains of the Galician Region (North-West of Spain). J. Dairy Res., 60: Ann. Microbiol., 53, (2003) 185

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