The influence of glycerol upon L. reuteri activity against enteropathogens

A influência do glicerol sobre a atividade de L. Reuteri contra enteropatógenos

Abstracts

BACKGROUND:

Probiotics containing L. reuteri are popular for treating and preventing bacterial gastrointestinal infections. L. reuteri, produces reuterin, an antibiotic that inhibits gram-negative bacteria. Reuterin production is the result of glycerol fermentation by L reuteri. Although L. reuteri is normally present in the gastrointestinal system, only small amounts of glycerol are usually available; therefore, the production of reuterin may not occur and this could reduce the effectiveness of the probiotic supplement. Our objective is to identify the minimum concentrations of glycerol required for L. reuteri to exert an inhibitory effect on enteropathogenic enterobacteriaceae.

METHOD:

Samples containing 10⁽⁸⁾ colony forming units (CFU) of L. reuteri DSM17938 (Colikids^®, Ache, Brazil/BioGaia, Sweden) were grown with varying concentrations of glycerol (0.05-5%). 10⁽⁶⁾ CFU of E.coli (CDC0126/INCQS/FIOCRUZ), Shigella flexneri (ATCC/120022), S. enterica (ATCC6539) and Y. enterocolitica (ATCC9610) were inoculated with L. reuteri in the different glycerol concentrations. Each enterobacteria and glycerol 5% without L. reuteri cultures were used as positive control groups.

RESULTS:

All bacteria were completely inhibited at higher ranges of glycerol concentrations (0.2-5%) and grew at lower concentrations (0.05-0.1%).

CONCLUSION:

L. reuteri requires at least 0.2% of glycerol to completely inhibit enterobacterial growth. These preliminary findings may influence the current method of use of probiotic supplements. The antibiotic activity of L. reuteri may have potential clinical use against important enteropathogens.

KEYWORDS:
L. reuteri; reuterin; enteropathogenic enterobacteriaceae; glycerol

CONTEXTO:

Os probióticos que contêm L. reuteri são populares para tratar e prevenir infecções bacterianas do trato gastrointestinal. L. reuteri produz reuterina, um antibiótico que inibe bactérias gram-negativas. A produção de reuterina depende da presença de quantidades adequadas de glicerol, cuja fermentação resulta na produção do antibiótico. Embora L. reuteri esteja normalmente presente no sistema GI, apenas pequenas quantidades de glicerol estão geralmente disponíveis; portanto, a produção de reuterina pode não ocorrer o que poderia reduzir a eficácia do suplemento probiótico. Nosso objetivo é identificar as concentrações mínimas de glicerol necessárias para que L. reuteri exerça um efeito inibitório nas enterobacteriaceas enteropatogênicas.

MÉTODO:

108 CFU de L. reuteri DSM17938 (Colikids^®, Ache, Brasil / BioGaia, Suécia) cresceu com concentrações variáveis de glicerol (0,05-5%). Foram inoculadas 106 UFC de E. coli (CDC0126 / INCQS / FIOCRUZ), Shigella flexneri (ATCC / 120022), S. enterica (ATCC6539) e Y. enterocolitica (ATCC9610) com L. reuteri nas diferentes concentrações de glicerol. Cada enterobacteria e glicerol 5% sem culturas de L. reuteri foram utilizadas como grupos de controle positivo.

RESULTADOS:

Todas as bactérias foram completamente inibidas em maiores concentrações de glicerol (0.2-5%) e cresceram em concentrações mais baixas (0.05-0.1%).

CONCLUSÃO:

L. reuteri requer pelo menos 0,2% de glicerol para inibir completamente o crescimento de enterobacteria. Essas descobertas preliminares podem influenciar o método atual de uso de suplementos de probióticos. A atividade antibiótica de L. reuteri pode ter potencial uso clínico contra importantes enteropatógenos.

PALAVRAS-CHAVE:
L. reuteri; reuterina; Enterobacteriaceae enteropatogênicas; glicerol

INTRODUCTION

Enteropathogenic Escherichia coli (EPEC), alongside Salmonella enterica, Shigella flexneri and Yersinia enterocolitica, can be dangerous, especially to communities with low access to proper hygiene and medical care, as they are associated with consumption of food that has been in contact with feces.¹ Enteropathogenic Escherichia coli (EPEC) may affect bottle-fed infants because of contaminated water used for rehydration,² and in India, EPEC is responsible for approximately 3% of the diarrheas in children.³ Unfortunately, the overuse of antibiotics has made several types of E. coli resistant, and among 162 diarrheagenic E. coli cultures, nearly 86% have been shown to be resistant to Ampicillin, 77% to Chloramphenicol, 30% to Cefuroxime, 24% to Cefotaxime, and a striking 88% to Trimethoprim-Sulfamethoxazole.⁴ Therefore, since common antibiotics are inefficient at inhibiting EPEC, alternative methods have been proposed. For instance, Lactobacillus reuteri (L. reuteri) can inhibit gram-positive and gram-negative bacteria by producing reuterin, an antibiotic substance created from the organism's fermentation of glycerol. Treatment with L. reuteri as a probiotic supplement has been shown to decrease diarrhea in children with mild gastroenteritis.⁵

However, L. reuteri is highly fastidious, and must have glycerol available for fermenting, in order to properly grow and produce reuterin. The human gastrointestinal tract, however, has low availability of glycerol, since most is enveloped in micelles.⁶ Although several studies show that L. reuteri can inhibit the growth of EPEC, what is unknown is whether growing EPEC with larger amounts of glycerol, the inhibitory power of L. reuteri will be improved.

The objective of this study is to identify the minimum concentrations of glycerol required for L. reuteri to exert an inhibitory effect on enteropathogenic entero-bacteriaceae.

MATERIALS AND METHODS

Prior to initiating studies with Shigella flexneri ATCC 120022, Salmonella enterica ATCC 6539 and Yersinia enterocolitica ATCC 9610, only L. reuteri DSM 17938 (equivalent to one drop of COLIKids^®, Ache, Guarulhos, Brazil & BioGaia, Stockholm, Sweden) was grown in 3mL of Brain Heart Infusion (BHI) broth with and without glycerol at a concentration of 15% to determine the influence of glycerol on its growth pattern in triplicate. The same was done for enteropathogenic Escherichia coli (EPEC) CDC0126 INCQS FIOCRUZ, which was grown with and without glycerol (15%). L.reuteri and EPEC were grown together with and without glycerol (15%) (Table 1).

Table 1
Initial results evaluating the influence of glycerol on the growth patterns of L. reuteri alone, EPEC alone and the combination of both L reuteri and EPEC.

N	With 15% glycerol (in CFU)			Without 15% glycerol (in CFU)
	EPEC	L. reuteri	L. reuteri+ EPEC.	EPEC	L. reuteri	L. reuteri+ EPEC.
1	106	105	2.8x101*	106	106	105*
2	106	106	0*	106	105	105*
3	105	105	0*	105	105	105*
Avg.	7.0x105	4.0x105	9.33*	7.0x105	3.67x105	105*
p-value						<0.001

• N = triplicate number; CFU = colony forming units; Avg. = average;
• ^*
  Count of EPEC CFU; p-value = L. reuteri + EPEC with glycerol versus L. reuteri + EPEC without glycerol

After the preliminary experiment, the second step was to incubate 10⁸ CFU of L. reuteri DSM 17938 with varying concentrations of glycerol (5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%) for 24 hours at 35ºC.

Then, 10⁶ CFU of each of Escherichia coli (EPEC) CDC0126 INCQS FIOCRUZ, Shigella flexneri ATCC 120022, Salmonella enterica ATCC 6539 and Yersinia enterocolitica ATCC 9610 were inoculated individually in the tubes containing L. reuteri in different glycerol concentrations, and incubated for 24h at 35ºC.

Finally, 10µL of the samples were sown on MacConkey Agar plates and grown for another 24h at 35ºC, and colonies were counted (measured in Colony Forming Units - CFU per milliliter). In tubes with BHI broth, each one of the enterobacteriae and glycerol at 5% without L. reuteri were used as positive control groups.

Temperature of all incubators was set at 35ºC. This study was conducted in a Biosafety Level 2 Cabinet.

The T-test was applied comparing the results in Table 1 for L. reuteri + EPEC with glycerol (15%) and L. reuteri + EPEC without glycerol. The T-test was not applied for any of the results in Table 2, since there was only one replicate of each trial.

Table 2
Colony Forming units/mL of Gram negative bacteria according to glycerol concentrations.

	CFU/mL of Gram Negative Bacteria
glycerol concentration (%)	5%	2%	1%	0.5%	0.2%	0.1%	0.05%
EPEC	0	0	0	0	0	5.3x102	5.7x102
S. flexneri	0	0	0	0	0	105	>105
S. enterica	0	0	0	0	0	105	>105
Y. enterocolitica	0	0	0	0	0	0	>105

• *EPEC: Enteropathogenic Escherichia coli

RESULTS

As shown in Table 1, when L. reuteri is grown with glycerol, EPEC is inhibited with only 9.3 CFU remaining. When it is not grown with glycerol, EPEC grows normally and 10⁵ CFU can be noted. There was a significant (p<0.001) difference in EPEC counts after glycerol (15%) was included with L. reuteri (Table 1). The inclusion of glycerol makes a significant difference in the inhibition of EPEC.

As shown in Table 2, when all gram-negative bacteria were studied under these conditions and increasing the content of glycerol, they showed the same pattern of inhibition, being completely inhibited at higher ranges of glycerol concentrations (0.2 to 5%) and growing at lower concentrations (0.1 to 0.05%) with the exception of Y. enterocolitica that only showed growth in glycerol 0.05%.

In all the control tubes, there were 10⁶ CFU of each enterobacteria.

DISCUSSION

L. reuteri requires at least 0.2% of glycerol to completely inhibit some enteropathogenic enterobacteria growths. Although studies show that reuterin produced by L. reuteri exerts an inhibition of bacterial activity,^7,8 in our study all enteropathogenic bacteria evaluated (EPEC, Shigella flexneri, Salmonella enterica and Yersinia enterocolitica) grew normally in the presence of L. reuteri. It was only when glycerol was added to this combination that L. reuteri completely inhibited these enteropathogens.

In the human body, concentration of glycerol is quite small. It would be essential to analyze how the manipulated amount of glycerol in our experiment contributed to the levels of inhibition, and what could be expected in the human body. Interestingly, glycerol concentrations ranging from 0.1 - 0.05% were not as efficient as amounts ranging from 0.2% and above. This demonstrates how the addition of glycerol to L. reuteri probiotic supplements might be necessary to have a more efficient inhibition of EPEC.

One limitation was that it would be more precise to have counted exactly the number of colonies in some samples (specifically ones that showed >10⁵ CFU). Another part of the experiment that could show abnormalities are the standard deviation values (SD), due to the fact that results were quantified as 10^6/5, and in these numbers, there is a large difference when calculating an average. However, having a result that is in the range of 10^6/5 indicates normal growth for healthy colonies, a point which SD does not account for.

As all data were collected with the same batch despite different times, it could be considered it would eliminate possible factors such as risking to having damaged or slightly different samples.

CONCLUSION

The consequences of the glycerol supplementation in bacterial pathogens of GI tract have yet to be fully explored, as the amount of glycerol available for adequate antimicrobial effect has not been studied. We are introducing a concept that requires further exploration as probiotics do indeed maintain health and good supplementation of natural microbiota.

Glycerol is essential for the antimicrobial activity of L. reuteri and reuterin production. These preliminary findings may influence the current method of use of probiotic supplements. It might be wise to consider using an additional dosage of glycerol when taking L. reuteri as a probiotic in order to guarantee a better result of probiotic supplements. Possibly if L. reuteri is taken as a probiotic with high glycerol concentrations, then enteropathogens could be inhibited. The antibiotic activity of L. reuteri may have potential clinical use against important enteropathogens.

Further work is needed concerning the production of reuterin and other metabolites that may alter probiotic effects and consequently the growth of enteropathogenic bacteria.

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Publication Dates

• Publication in this collection
  Nov-Dec 2017

History

• Received
  12 Oct 2017
• Reviewed
  13 Nov 2017
• Accepted
  11 Dec 2017