Glyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum

doi:10.1016/j.anaerobe.2013.01.005

Anaerobe

Volume 20, April 2013, Pages 74-78

https://doi.org/10.1016/j.anaerobe.2013.01.005 Get rights and content

Abstract

During the last 10–15 years, an increase of Clostridium botulinum associated diseases in cattle has been observed in Germany. The reason for this development is currently unknown. The normal intestinal microflora is a critical factor in preventing intestinal colonisation by C. botulinum as shown in the mouse model of infant botulism. Numerous bacteria in the gastro-intestinal tract (GIT) produce bacteriocines directed against C. botulinum and other pathogens: Lactic acid producing bacteria (LAB) such as lactobacilli, lactococci and enterococci, generate bacteriocines that are effective against Clostridium spp. A reduction of LAB in the GIT microbiota by ingestion of strong biocides like glyphosate could be an explanation for the observed increase in levels of C. botulinum associated diseases. In the present paper, we report on the toxicity of glyphosate to the most prevalent Enterococcus spp. in the GIT. Ingestion of this herbicide could be a significant predisposing factor that is associated with the increase in C. botulinum mediated diseases in cattle.

Introduction

Clostridium botulinum associated diseases in cattle appear in two forms, acute or chronic. The acute form of botulism occurs after the uptake of preformed botulinum neurotoxin (BoNT) in feeds causing flaccid paralysis and death by respiratory failure. Graham and Schwarze, [1] described the acute form of botulism as developing of paralysis without previous symptoms, leading to sudden death or slow recovery after complete muscle relaxation. In contrast, the chronic form of the disease is characterized by weakness, local paralysis, emaciation, muscular stiffness and recumbency of varying degrees. A neurologic manifestation may be highly aggressive behaviour resulting in damage to feeding troughs, mangers or fences. Animals may also display visual disturbances [1]. The causal bacterium, C. botulinum, is an ubiquitous Gram-positive, spore-forming obligate anaerobic bacterium. Strains generate neurotoxins that block the release of acetylcholine at the neuromuscular junctions. Amino acid variation within the BoNT results in seven immunologically distinct BoNT serotypes (A–G) which are further divided into subtypes [2], [3], [4], [5]. Two other bacteria, C. baratii and C. butyricum, can also produce BoNT F and E, respectively. The factors that determine the severity of the disease and the factors that prevent intestinal colonisation by C. botulinum spores are incompletely characterized [6]. In the mouse model of infant botulism, the normal intestinal microflora has been shown to be a critical factor in preventing intestinal colonisation by C. botulinum [7]. Wang and Sugiyama [8] reported that mice developed symptoms of botulism when they were treated with metronidazole before oral application of C. botulinum A and B spores. Some mice harboured relatively large amounts of toxin in the large bowel without displaying any clinical signs, possibly bound to phospholipids and gangliosides [9]. This is in line with other intestinal diseases, where the normal enteric microflora has been found to protect against colonization by a variety of bacterial pathogens [6]. The microflora of the GIT of domestic animals consists of a balanced composition of facultative and obligatory anaerobic bacteria. The mature microbiota profile varies considerably along the length of the GIT and may be specific to animal species and individuals [10].

Numerous bacteria of the GIT produce bacteriocines directed against some other bacterial species including pathogens. Among others, lactic acid bacteria like lactobacilli, lactococci and enterococci may generate such bacteriocines. Specifically, Enterococcus faecalis (E. faecalis) may generate an enterocin 1146 that was shown to be very effective against C. butyricum and C. perfringens [11], [12], [13], [14]. Glyphosate, N-(phosphonomethyl) glycine, the most widely used herbicide in the world, is the active ingredient in Roundup^® and contains also adjuvants such as polyethoxylated tallowamine [15]. Glyphosate is a strong systemic metal chelator and was initially patented for that purpose in 1964 [16]. It is also a selective and potent, microbiocide. Its herbicidal action is generated by chelating manganese required in the reduction of the FMN co-factor for the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase enzyme in the shikimate pathway, inhibiting this metabolic pathway of plants and also many microorganisms [17]. The extensive use of glyphosate as a broad-spectrum herbicide in agriculture, and especially the direct application of glyphosate to Roundup Ready^® soya, corn, rapeseed, cotton, sugar beets, and alfalfa fed to animals, leads to incorporation of residual glyphosate into the GIT. Moreover, glyphosate showed differences in sensitivity between micro-organisms [18], [19] which could disturb the normal gut bacterial community. The aim of this paper is to document the inhibitory effect of glyphosate on Enterococcus spp. which antagonises C. botulinum.

Section snippets

Isolation and identification of Enterococcus strains

Enterococcal isolates were isolated from cattle, horses, and algae (Chlorella vulgaris, (Ökologische Produkte Altmark Co, Germany) by plating specimens on citrate- acid-tween-carbonate (CATC agar, Oxoid, Germany) and incubated aerobically at 37 °C for 48 h. Typical red colonies were sub-cultured on Caso agar (3.5% Casein-Soya, 0.3% yeast extract, 0.1% glucose, 1.5% Agar Agar). These colonies were examined for Gram reaction and cellular morphology. All Gram positive, catalase-negative cocci

Results

All tested Enterococcus spp, isolated from Chlorella vulgaris and from faeces of cattle and horses (Table 1) inhibited neurotoxin production by C. botulinum types A, B, D and E reference strains. C. botulinum type C did not produce neurotoxin. Likewise, all enterococci co-cultivated with C. botulinum reduced the growth (cell numbers) of botulinum type A, B, C, D and E (Table 2). Even low numbers of E. faecalis (Fig. 1A) and E. faecium (Fig. 1B) inhibited BoNT production by all C. botulinum

Discussion

Antagonism between C. botulinum and bacterial members of the micro-ecosystem are well known [6], [15]. Special strains of C. perfringens, C. sporogenes and Bacillus cereus were able to antagonize C. botulinum Types A, B, F and E [21]. The growth of non-proteolytic strains of C. botulinum (Type 17B, Beluga and 202 F) was reduced by Lactobacillus spp., Lactococcus spp., Streptococcus spp. and Pediococcus spp. [22]. Okereke and Montville [23] reported that bacteriocine producers of the lactate

References (32)

K. Tsukamoto et al.
Binding of Clostridium botulinum type C and D neurotoxins to ganglioside and phospholipid. Novel insights into the receptor for clostridial neurotoxin
J Biol Chem
(2005)
E. Parente et al.
Characterization of Enterocin 1146, a bacteriocin from Enterococcus faecium inhibitory to Listeria monocytogenes
J Food Protect
(1992)
E. Parente et al.
Inhibition of Listeria in buffer, broth, and milk by Enterocin 1146, a bacteriocin produced by Enterococcus faecium
J Food Protect
(1992)
A. Schulz et al.
Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate
FEMS Microbiol Lett
(1985)
D.A. Wardle et al.
The influence of the herbicide glyphosate on interspecific interactions between four fungal species
Mycol Res
(1992)
M. Krüger et al.
Visceral botulism at dairy farms in Schleswig Holstein, Germany- Prevalence of Clostridium botulinum in feces of cows, in animal feeds, in feces of the farmers, and in house dust
Anaerob
(2012)
S. Rodgers et al.
Inhibition of nonproteolytic Clostridium botulinum with lactic acid bacteria and their bacteriocins at refrigeration temperatures
J Food Prot
(2003)
P. Peruzzo et al.
Levels of glyphosate in surface waters, sediments and soils associated with direct sowing soybean cultivation in north pampasic region of Argentina
Environ Pollut
(2008)
R. Graham et al.
Botulism in cattle
J Bact
(1921)
A. Goonetilleke et al.
Clostridial neurotoxins
J Neurol Neurosurg Psychiatry
(2004)

L.L. Simpson

Identification of the major steps in botulinum toxin action

Annu Rev Pharmacol Toxicol

(2004)

T.J. Smith et al.

Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids

PLoS ONE

(2007)

M.R. Popoff et al.

Clostridial toxins

Future Microbiol

(2009)

D.H. Burr et al.

Susceptibility to enteric botulinum colonization of antibiotic treated adult mice

Infect Immun

(1982)

N.M. Sullivan et al.

Inhibition of growth of Clostridium botulinum by intestinal microflora isolated from healthy infants

Microb Ecol Health Dis

(1988)

Y. Wang et al.

Botulism in metronidazole-treated conventional adult mice challenged orogastrically with spores of Clostridium botulinum type A or B

Infect Immun

(1984)

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Pathogenesis and toxinsGlyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum

Abstract

Introduction

Section snippets

Isolation and identification of Enterococcus strains

Results

Discussion

J Biol Chem

J Food Protect

J Food Protect

FEMS Microbiol Lett

Mycol Res

Anaerob

J Food Prot

Environ Pollut

Botulism in cattle

J Bact

Clostridial neurotoxins

J Neurol Neurosurg Psychiatry

Identification of the major steps in botulinum toxin action

Annu Rev Pharmacol Toxicol

Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids

PLoS ONE

Clostridial toxins

Future Microbiol

Susceptibility to enteric botulinum colonization of antibiotic treated adult mice

Infect Immun

Inhibition of growth of Clostridium botulinum by intestinal microflora isolated from healthy infants

Microb Ecol Health Dis

Botulism in metronidazole-treated conventional adult mice challenged orogastrically with spores of Clostridium botulinum type A or B

Infect Immun

Pathogenesis and toxins
Glyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum