Journal of Applied Bacteriology 1992, 73, 480-483

In vitro studies on the inhibition of the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus Maria C. Apella, Sllvla N. Gonzalez, Maria E. Nader de Madas, Nora Romero and G. Oliver Centro de Referencia para Lactobacilos (CERELA) , Chacabuco 145, Tucumhn, Argentina 4027/10/91: accepted 3 June 1992

M.C. APELLA. S.N. GONZALEZ, M.E. NADER DE MACIAS, N. ROMERO A N D G . OLIVER. 1992. T h e inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. T h e effect was not due to p H alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. T h e higher inhibition was produced by the mixture of lactobacilli (35-5 2.5% at 6 h culture, 57.4 f 1.9% at 9 h and 91.2 f 1.2% at 14 h). T h e degree of inhibition was higher when the relationship pathogen : lactobacilli was 1 : lo3. The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37°C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of S h . sonnei.

INTRODUCTION

Dysentery and diarrhoea caused by shigellas are major public health problems in developing countries (Anon. 1986). Shigella sonnei and Sh. Jexneri are the most frequently identified pathogens in children with enteric disease ranging from a mild diarrhoea to a severe dysenteric syndrome with blood, mucus and pus in stools. Endemic shigellosis caused by these organisms has a high morbidity rate, occasionally with substantial mortality. Lactic acid bacteria are used as starters in several dairy products and other fermented foods (Lawrence et al. 1976). They are used to inhibit the growth and activity of several micro-organisms (Fernindez et al. 1982). Two strains of lactobacilli, Lactobacillus casei and Lact. acidophilus, were isolated from faeces of healthy children in our laboratory. These strains were able to survive in gastric juice and bile and had an inhibitory effect on the growth of enteropathogenic micro-organisms such as Escherichia coli. Shigellosis is the most communicable of all bacterial enteric diseases since as few as 10 live organisms can cause disease (Fernindez et al. 1982; Anon. 1986). The purpose of this work was to examine the inhibitory effect of Lact. Correspondence to :Dr Guillermo Oliver, Centro de Referencia para Lactobacilos ( C E R E L A ) , Chacabuco 145, 4000 Tucuman, Argentina

casei and Lact. acidophilus on the growth of S h . sonnei under different experimental conditions. We report the effect of a fermented milk containing Lact. casei and Lact. acidophilus on S h . sonnei. This may have a considerable potential for the treatment of infantile diarrhoea. MATERIALS AND METHODS Mlcro-organisms

The bacteria used were Lact. acidophilus and Lact. casei, isolated from faeces of healthy children and identified according to the method of Kandler & Weiss (1985). Shigella sonnei was isolated from a human stool and was identified by biochemical and serological tests (Rowe & Gross 1984). Culture media

The following media were used to grow the lactobacilli: LAPTg (Raibaud et al. 1961; containing (g/l): yeast extract, 10; peptone, 15; tryptone, 10; glucose, 10; Tween 80, 10); MRS medium (Oxoid; De Man et al. 1960); LBS (lactobacillus selective medium; Rogosa et al. 1951); and 10% (w/v) skim milk powder. BHI (Brain Heart Infusion: Difco) was used to grow the shigella.

SHIGELLA INHIBITION B Y L A C T O B A C I L L I : IN VlTRO

Culture methods

Lyophilized stock cultures of lactobacilli were transferred to LAPTg broth, incubated at 37°C and then subcultured in the same media until an active culture was obtained. The strains were maintained by weekly transfer in the same media or monthly in milk with 1% yeast extract. Shigella sonnei was grown and maintained in BHI. For the mixed cultures, the organisms were subcultured three times in LAPTg broth. The virulence of Sh. sonnei was tested by its capability to produce red colonies in media containing Congo red dye, according to Payne & Finkelstein (1977), and by the Sereny Test (Sereny 1957). Determination of antagonistic activity

The broth media were inoculated with 1% (v/v) of a ‘hold’ culture of lactobacilli, incubated at 37°C for 48 h and the cells harvested by centrifugation at 10000 g for 15 min. The supernatant fluid was then filtered through Millipore membranes (0.25 pm). The antagonistic activity of the filtered supernatant fluid and that of the pellet resuspended in 0.5 ml of distilled water was tested by different methods: (1) Paper disc assay : a lawn of Sh. sonnei was prepared by spreading 0.1 ml of an active culture (about 9 x lo4 cfu) on the surface of LAPTg agar and Bacto MacConkey agar (Difco) in 12 cm diameter Petri dishes. Paper discs (1/4 inch; Difco) were dipped into the supernatant fluid or into the pellet cell suspension and placed on the lawn. After a few minutes at room temperature, to allow diffusion, the plates were incubated at 37°C for 24-48 h and the diameter of the inhibition rings measured. (2) The same media was stabilized at 45°C and then inoculated with 0.1 ml of the Sh. sonnei culture, stirred gently and poured into Petri dishes. Holes 8 mm in diameter were punched in the agar and then filled with the samples. The plates were kept at room temperature until the liquid was absorbed, then incubated and the inhibition rings measured. (3) Deferred inhibition assay (Tagg et al. 1976): LAPTg agar medium stabilized at 45°C was inoculated with 1 x 10’ and 1 x lo3 cfu of an active culture of the lactobacillus strains and poured into Petri dishes. One group of dishes was incubated at 37°C for 24 h and then a layer of semi-solid agar (0.75%) of about 8 mm thick was added, followed by another layer of agar inoculated with 1 x 10’ cfu of Sh. sonnei. The plates were incubated at 37°C for 24 h. The second group of plates was prepared in the same way, but without incubation before the addition of the second and third layer. Growth of mixed cultures In iiquld medium

Flasks containing LAPTg broth were inoculated with either Lact. casei, Lact. acidophilus or a mixture of both. In these

481

experiments, the initial lactobacilli concentration was about lo7 cfu/ml. After incubation at 37°C for 12 h the flasks were inoculated with Sh. sonnei (lo7 cfu/ml) and incubated again at the same temperature for 12 h. This was repeated with a second group of flasks, except that the microorganisms were inoculated at once (pathogens in a lo7 or lo3 cfu/ml and lactobacilli at a lo7 cfu/ml concentration) and omitting the first period of incubation. For the quantitative determination of the surviving micro-organisms, dilutions of the cultures in 0.1% (w/v) peptone solution were plated in duplicate on LBS medium for lactobacilli and MacConkey agar for the shigella. The plates were incubated at 37°C for 24-48 h in a C 0 2 atmosphere. The Student’s test was used to determine the arithmetic standard deviation of results and their significant differences.

RESULTS

The supernatant fluids and pellets of the lactobacilli tested by the paper disc and the punched hole methods showed inhibition of growth of Sh. sonnei. The inhibitory effect was variable, however, depending on the grown medium, and decreased in the order: LAPTg > NFM > LBS > MRS (Table 1). The deferred method showed that inhibition by Lact. casei or Lact. acidophilus took place only when there was a period of incubation before the inoculation of Sh. sonnei. Preincubation was not necessary, however, when the mixture of lactobacilli was used. When it was grown in pure culture in liquid media, Sh. sonnei had an exponential phase of between 4-6 h, reaching the stationary phase after approximately 18 h. In a mixed culture, however, the exponential phase was markedly shortened (Fig. 1).

Table 1 In vitro inhibition of Shigella sonnei by lactobacilli grown in different media by the disc assay method

Shigella inhibition Lactobacilli grown in : Lactobacilli tested

LAPTg

MRS

Lactobacillus casei Lact. acidophilus Lact. casei Lact. acidophilus

22.0 18.0 22.0

+

LBS

NFM

8.0

11.0

11.5 12.0

12.5

13.0 14-0 13.5

13.5

The values represent the diameter of inhibition zones in mm. The pathogen was inoculated at lo5 cfu/plate and lactobacilli impregnated at lo’ cfu/disc. Controls were performed with shigella seeded in all the media tested.

482 M A R ~ Ac. APELLA E r A L .

51 4 10

20

24

tI

3

Time ( h )

Fig. 1 Kinetics growth of mixed culture of Shigella sonnei and lactobacilli. The cultures were grown in LAPTg broth at 37°C. 0 ,Sh. sonnei (control); 0, Sh. sonnei Lact. casei; 0 ,Sh. sonnei Lact. acidophilus; A, Sh. sonnei Lact. casei Lact. acidophilus

+

+

+

0 of Shigella I

I

I

I

I

I

4

8

12

16

20

24

Time ( h )

+

Calculation of the inhibition of S h . sonnei in percentage terms (Table 2) shows that it was most marked in the mixture of lactobacilli (P< 0.01). A comparison of the inhibition percentage produced with different pathogen : lactobacilli relationships showed that there was greatest inhibition with the mixture of lactobacilli in the proportion lo7 lactobacilli : lo4 S h . sonnei. The percentage of shigella inhibition obtained at 6 h was 43.4% with Lact. casei, 34.9% with Lact. acidophilus and 70% with the mixture of both lactobacilli. Incubation of pure cultures of the lactobacilli for 12 h, as well as in mixed culture, before inoculation of S h . sonnei, prevented the growth of the latter (Fig. 2).

One interesting observation was that growth rates of Lact. acidophilus, Lact. casei and S h . sonnei in mixed cultures were higher than those with pure cultures (Table 3).

DISCUSSION T h e inhibitory effect of the lactobacilli on the growth of S h . sonnei was not due to a lowering of p H nor to the action of acids normally produced by lactobacilli, because the

Table 2 Inhibition of Shigella sonnei

Time (h) Associative cultures

6

9

14

Sh. sonnei + Lact. casei Sh. sonnei + Lact. acidophilus Sh. sonnei + Lact. casei + Lact . acidophilus

18.7 f 2.8 25.8 f 2.1 35.5 f 2.5

29.2 f 3.2 46.4 2.8 57.4 f 1.9

84.2 f 1.9 86.5 f 3.3 91.2 f 1.6

The relationship pathogen : lactobacilli was 1 : 1.

.,

Fig. 2 Kinetics growth of mixed culture of Shigella sonnei and lactobacilli. The lactobacilli were grown at 37°C for 12 h and Pure Lact. casei; shigella inoculated at this time. 0, lactobacilli mixture; 0 ,pure Lact. acidophilus; A,pure Sh. sonnei; 0, shigella in presence of Lact. casei; 0 ,shigella in presence of mixture of lactobacilli; A, shigella in presence of Lact. acidophilus

presented as percentage obtained during growth at different times

SHIGELLA INHIBITION BY LACTOBACILLI: IN VITRO

Table 3 Growth rates of Lactobacillus casei and Lactobacillus acidophilus in pure and in mixed culture with Shigella sonnei

Pure culture Mixed culture with Sh. sonnei

Lact. casei

Lact. acidophilus

0.62 0.83

0-45 1.06

The values are expressed as h- and taken at 6 h of culture. addition of hydrochloric, lactic or acetic acids to the culture media, in amounts necessary to bring the p H to 5-6, did not impede the normal growth of Sh. sonnei. Another possible reason for inhibition, competition for nutrients, may also be discarded, because they are in excess of the media, whereas the specific growth rate of Sh. sonnei in pure culture was higher than those of the lactobacilli (Table 4). Inhibition also took place through a layer of agar, indicating that the inhibitory substances are extracellular and diffusible. Such substances might be peptidic, such as antibiotics, lactocidines and acidolines, which are produced by lactobacilli and are active against many Gram-negative microorganisms (Tagg et al. 1976). The production of these antibiotics is influenced by medium composition, which might account for the unequal inhibitory effect observed when different media were used. Further studies are needed to determine the exact nature of the inhibitory substance(s). T h e necessity for a preincubation period to obtain inhibition by single strains of lactobacilli means that there was a critical concentration of inhibitory substances below which Sh. sonnei was able to grow. Furthermore, it was shown that the inhibitory substances are normal products of the metabolic activity of lactobacilli, as there was no necessity for the presence of Sh. sonnei to mgger their synthesis. Moreover, the inhibition produced by mixed cultures of the lactobacilli, without preincubation, indicates the possibility of a higher rate of production of the inhibitory substances than by single strains. I t may be that the enhanced inhibitory capacity was the result of.a synergistic effect due to positive interactions of several inhibitory substances. This was also observed when the inhibition produced by the mixture of lactobacilli was higher than that obtained individually, irrespective of the shigella : lactobacilli ratios (1 : 1 (lo' cfu of both) or 1 : lo3 (lo4 cfu pathogen : lo7 cfu lactobacilli)). This last experiTable 4 Growth rates of Shigella sonnei in pure and mixed

culture

+

+

+

Shigella

Sh. Lact. casei

Sh. Lact. acidophilus

Sh. Lact. casei iLact. acidophilus

1.52

1.85

1.78

1.64

The values are expressed as h - I and taken at 6 h of culture.

483

ment was carried out to reproduce the natural environment of the gut, where there are fewer pathogens and large numbers of lactobacilli when fermented milk is consumed. Several studies have shown that lactobacilli constitute one of the dominant groups of intestinal and faecal organisms being at levels between lo7 and 10" (Shahani & Ayebo 1980). Gonzilez et al. (1990) have shown that lactobacilli are present in faeces at levels of lo5 and 108/g when fermented milk is consumed. These must be 10 to 100 shigellas to produce a clinically-apparent infection or to produce intestinal colonization in humans (Fernindez et al.

1982). This work shows that Sh. sonnei is inhibited by lactobacilli used in the preparation of fermented milk.

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