Current Microbiology
© Springer Science+Business Media New York 2012
10.1007/s00284-012-0277-2

The Effect of Glyphosate on Potential Pathogens and Beneficial Members of Poultry Microbiota In Vitro

Awad A. Shehata1, 2, 3  , Wieland Schrödl1, Alaa. A. Aldin4, Hafez M. Hafez5 and Monika Krüger1
(1)
Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germany
(2)
Albrecht Daniel Thaer-Institute of Agronomy, University Leipzig, Leipzig, Germany
(3)
Avian and Rabbit Disease Department, Faculty of Veterinary Medicine, Menoufiya University, Sadat City, Egypt
(4)
Institute of Parasitology, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany
(5)
Institute of Poultry Disease, Free University, Berlin, Germany
 
 
Awad A. Shehata
Received: 14 August 2012Accepted: 15 November 2012Published online: 9 December 2012
Abstract
The use of glyphosate modifies the environment which stresses the living microorganisms. The aim of the present study was to determine the real impact of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. The presented results evidence that the highly pathogenic bacteria as Salmonella Entritidis, Salmonella Gallinarum, Salmonella Typhimurium, Clostridium perfringens and Clostridium botulinum are highly resistant to glyphosate. However, most of beneficial bacteria as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacterium adolescentis and Lactobacillus spp. were found to be moderate to highly susceptible. Also Campylobacter spp. were found to be susceptible to glyphosate. A reduction of beneficial bacteria in the gastrointestinal tract microbiota by ingestion of glyphosate could disturb the normal gut bacterial community. Also, the toxicity of glyphosate to the most prevalent Enterococcus spp. could be a significant predisposing factor that is associated with the increase in C. botulinum-mediated diseases by suppressing the antagonistic effect of these bacteria on clostridia.

Introduction

Glyphosate (N-(phosphonomethyl) glycine) is a highly effective herbicide because of its potent and specific inhibition of 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS), an enzyme of the shikimate pathway that governs the synthesis of aromatic amino compounds in higher plants, algae, bacteria and fungi [2]. Its herbicidal action is generated by chelating manganese required in the reduction of the flavin mononucleotide (FMN) co-factor EPSPS [12]. It is possible that glyphosate can spread in the ecosystem and reach plants, animals and food chain. Glyphosate and its metabolite aminomethylphosphonate (AMPA) could be detected in green immature seed [33], harvested seeds [16] and in ground water [47]. Glyphosate and AMPA are amongst the first major pollutants of surface waters [25]. In the literature, different opinions exist about the safety of this herbicide. Some reports have not found any significant risk to human and animal health by the use of glyphosate, since this EPSPS enzyme is absent in animals [15, 52]. However, other inhibition pathways are involved, like cytochrom P450 aromatase inhibition. It has also been shown that glyphosate in vertebrates is genotoxic [44] and teratogenic [40]. Glyphosate is cytotoxic to human placental, embryonic kidney and liver cell lines at very low sub-agricultural dilutions [4, 5, 20]. In vivo, the adverse effects of glyphosate on male reproductive system of ducks and rabbits [39, 59] as well as liver of rats [6, 7] were reported. The use of such herbicides modifies the environment which stresses the living organisms [35, 55]. In addition, they can disturb also the fresh water microbial communities directly or indirectly and reduces the biodiversity of species in the aquatic community [46]. Moreover, glyphosate showed differences in sensitivity between microorganisms [9, 13, 23]. Normal enteric microbiota has been found to protect the gastrointestinal tract (GIT) against colonisation by a variety of pathogens [11]. The microbiota 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 [22]. Numerous bacteria of the GIT produce bacteriocines directed against some other bacterial species including pathogens. Lactic acid bacteria like lactobacilli, lactococci and enterococci may generate such bacteriocins, specifically; Enterococcus faecalis may generate an enterocin 1146 that was shown to be very effective against Clostridium butyricum and Clostridium perfringens [28, 41, 42]. In the present study, the effect of glyphosate on growth and viability of potential pathogens and beneficial members of poultry microbiota were investigated in vitro.

Materials and Methods

Glyphosate

Roundup UltraMax® (Monsanto, USA) which contains 450 mg/ml of glyphosate was used in this study.

Strains and Cultural Conditions

Experiments were carried out with Bacillus badius, Bacillus cereus, Bacteriodes vulgatus, Bifidobacterium adolescentis, Campylobacter coli, Campylobacter jejuni, C. perfringens, C. botulinum type A, C. botulinum type B, Escherichia coli, E. coli 1917 strain Nissle, E. faecalis, E. faecium, Lactobacillus buchneri, L. casei, L. harbinensis, Riemerella anatipestifer, Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Typhimurium, Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus lentus were used. Cultural conditions and origin of these strains are described in Table 1.
Table 1
Target strains used, their origin and medium used for glyphosate experiments
Genus/species
Origin
Reference no. and related collections
Lab. no.
Culture media
Incubation conditions
Agar
broth
Bacillus badius
Green algae (Altmark Co, Germany)
Isolated in this study
1/12
Blood agara
RCMb
37 °C/24 h aerobic
Bacillus cereus
Green algae (Altmark Co, Germany)
Isolated in this study
2/12
Blood agar
RCM
37 °C/24 h aerobic
Bacteriodes vulgatus
Chickens
Isolated in this study
3/12
Blood agar
RCM
37 °C/48 h anaerobic
Bifidobacterium adolescentis
Chickens
Isolated in this study
4/12
MRSc
MRS
37 °C/48 h anaerobic
Institute of Bacteriology, Leipzig University
11151/03
11151/03
Caso-modified agar
Caso-modified broth
37 °C/48 microaerophilic
Campylobacter jejuni
Institute of Bacteriology, Leipzig University
188
188
Caso-modified agar
Caso-modified broth
37 °C/48 microaerophilic
C. perfringens
RIPAC-LABOR GmbH, Germany
884/2
884/2
NeoPd
RCM
37 °C/24 h anaerobic
C. botulinum type A
National collection of type culture (NCTC)
7272
7272
Blood agar
RCM
37 °C/48 h anaerobic
C. botulinum type B
National collection of type culture (NCTC)
7273
7273
Blood agar
RCM
37 °C/48 h anaerobic
E.coli
Chickens
Isolated in this study
5/12
Blood agar
RCM
37 °C/24 h aerobic
E. coli 1917 strain Nissle
Ardeypharm GmbH, Herdecke, Germany
Nissle
Nissle
Blood agar
RCM
37 °C/24 h aerobic
Enterococcus faecalis
Chlorella vulgaris
Isolated in this study
6/12
CATCe
RCM
37 °C/24 h aerobic
Enterococcus faecium
Chickens
Iisolated in this study
7/12
CATC
RCM
37 °C/24 h aerobic
Lactobacillus buchneri
Effective microorganisms (Multikraft Co, Austria)
12/25
12/25
MRS
MRS
37 °C/48 h anaerobic
Lactobacillus casei
Effective microorganisms (Multikraft Co, Austria)
12/26
12/26
MRS
MRS
37 °C/48 h anaerobic
Lactobacillus harbinensis
Chickens
Isolated in this study
8/12
MRS
MRS
37 °C/48 h anaerobic
Riemerella anatipestifer
Institute of Poultry Diseases, Berlin University
12109
12109
Blood agar
RCM
37 °C/24 h aerobic
Salmonella Enteritidis
Chickens
Isolated in this study
9/12
Caso agarf
RCM
37 °C/24 h aerobic
Salmonella Gallinarum
Institute of Poultry Diseases, Berlin University
Z34/11
Z34/11
Caso agar
RCM
37 °C/24 h aerobic
Salmonella Typhimurium
Chickens
Isolated in this study
10/12
Caso agar
RCM
37 °C/24 h aerobic
Staphylococcus aureus
Chickens
Isolated in this study
11/12
Blood agar
RCM
37 °C/24 h aerobic
Staphylococcus haemolyticus
Chickens
Isolated in this study
12/12
Blood agar
RCM
37 °C/24 h aerobic
Staphylococcus lentus
Chickens
Iisolated in this study
13/12
Blood agar
RCM
37 °C/24 h aerobic
aBlood agar (Fluka, Germany)
bReinforced clostridial medium (RCM, Sifin, Germany)
cdeMan, Rogosa and Sharpe Lactobacillus Agar (Oxoid, Germany)
dNeomycin–polymixin blood agar (neomycin (100 mg/l, Roth, Germany)/polymyxin B (50 mg/l Fluka, Germany)
eCitrat-Azid-Tween-Carbonat agar (Oxoid, Germany)
fCaso agar (3.5 % casein-soya, 0.3 % yeast extract, 0.1 % glucose, 1.5 % agar)

Bacterial Identification

Bacterial strains were tested using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF). In brief, about 10 mg of cell material of the cultured strains were suspended in 300 μl of sterile water. 900 μl of absolute ethanol was added, and the mixture was centrifuged at 10,000 rpm for 2 min. The supernatant was discarded, and the pellet was suspended in 50 μl formic acid (70 % v/v). After adding 50 μl acetonitrile (AN), the mixture was centrifuged at 10,000 rpm for 2 min. 1 μl of the clear supernatant was transferred to the MALDI target and allowed to dry. A saturated solution of α-cyano-hydroxy-cinnaminic acid (Bruker Daltonik GmbH) in a standard organic solvent mixture (2.5 % trifluoroacetic acid to AN 50 % in water was added to resuspended 1 μl of the dried material. All chemicals used were of the highest quality (Merck, designated to be especially suitable for HPLC or MALDI-based techniques). Before each MALDI run, E. coli 1917 strain Nissle was analysed to serve as the positive control and calibration standard. The MALDI–TOF MS analysis was performed using a Bruker microflex LT mass spectrometer (Bruker Daltonik Co), and the spectra were automatically identified using the Bruker BioTyper™ 1.1 software. C. perfringens was identified by MALDI-TOF and multiplex PCR [3], data are not shown.

Effect of Glyphosate on Different Bacteria

The minimal inhibitory concentration (MIC) of Roundup UltraMax® was determined in triplicate in a 24-well microtiter plate. 100 μl of tested bacteria (10cfu/ml) was added to 900 μl broth media containing different concentrations of glyphosate (5.0, 2.40, 1.20, 0.60, 0.30, 0.15 and 0.075 mg/ml). Plates containing diluted glyphosate and bacteria were incubated at 37 °C (Table 1). Bacterial growth was evaluated on suitable agar medium. Culture condition for each bacterium is shown in Table 1. The MIC value was evaluated by quantitative analysis of bacteria on agar plate. The morphology of bacteria was examined microscopically.

Effect of Glyphosate on C. botulinum Type A and B

Clostridium botulinum was cultured anaerobically in a cooked meat broth at 37 °C for 5 days, followed by cultivation in reinforced clostridial medium (RCM, Sifin, Germany) anaerobically at 37 °C for 3 days. C. botulinum types A and B were heated at 80 °C for 10 min and left at room temperature under aerobic condition. Cultures were tested daily for sporulation using a Gram or Rakette stain. To study the effect of glyphosate on C. botulinum strains, heat treated spores or vegetative cells were added to RCM medium at a final concentration of 104 cfu/ml. The inhibitory effect of glyphosate was determined using the following concentrations: 5, 2.40, 1.20, 0.60, 0.30, 0.15 and 0.075 mg/ml. The mixture was incubated anaerobically at 37 °C for 5 days. C. botulinum was quantified using the most probable number (MPN) estimation method. C. botulinum type A and B neurotoxins (BotNT) were analysed using ELISA [30]. In brief, ELISA was performed in flat-bottomed ELISA plates (96 wells, high binding; Costar, Corning, New York, USA). All standard volumes were 100 μl/well and the standard incubation condition was 1 h at room temperature (1 h at RT) on a microtitre plate shaker (400 rpm). The coating buffer was 0.1 M NaHCO3 and the wash solution (WS) was 0.9 % NaCl with 0.1 % Tween 20 (Sigma-Aldrich, Taufkirchen, Germany). After coating the wells with capture antibodies (3 μg ml, rabbit IgG against C. botulinum neurotoxin type A or B, Institute of Bacteriology and Mycology, University of Leipzig), the plates were incubated overnight at 4–6 °C. ELISA plates were washed twice with WS and loaded with the diluted specimens. Supernatants of the cultures were diluted 1:10 or higher in assay buffer (assay buffer: 20 mM Tris, pH 8.00 [adjusted with 1 M HCl], 0.9 % NaCl, 5 mM EDTA, 1.0 % gelatine from cold water fish skin, 0.2 % bovine serum albumin, 0.1 % rabbit IgG and 0.2 % Tween 20 (all from Sigma-Aldrich or Fluka, Taufkirchen, Germany). The plates were incubated for 1 h at RT, washed five times with WS and loaded with the detection antibodies conjugated with horseradish peroxidase, diluted in assay buffer. BotNT types A and B were detected with 2.5 μg/ml horse ([Fab]2 from IgG) against C. botulinum types A and B, respectively (Novartis Vaccines and Diagnostics GmbH, Marburg, Germany). After 1 h incubation at RT, the plates were washed four times with WS. All washing steps were done by a Nunc-Immuno-Washer 12 (Nunc, Wiesbaden, Germany). The antibody bound marker enzyme, horseradish peroxidase, was detected by adding 3 mM H2O2 and 1 mM 3,3′,5,5′-tetramethylbenzidine (TMB) in 0.2 M citrate-buffer to each well (pH 4.0). The substrate reaction was stopped with 1 M H2SO4 (50 μl/well). The optical density (OD) was measured using a microplate ELISA reader at 450 nm. The supernatant with known concentrations of the BotNT types A and B (Institute of Bacteriology and Mycology, University of Leipzig) were used as a standard.

Effect of Glyphosate on Campylobacter spp.

The effect of glyphosate on C. coli and C. jejuni was tested in a 10 ml tissue culture flask in Caso-modified broth media containing 3.5 % caso broth 0.3 % yeast extract, 0.3 % casein hydrolysate, 0.03 % FeSO4 and 0.03 % natrium pyruvate. Glyphosate was added with different concentrations (5.0, 2.40, 1.20, 0.60, 0.30 and 0.15 mg/ml). Tested bacteria was added at a final concentration of 104 cfu/ml under microaerophilic conditions at 37 °C for 48 h. Bacterial growth in the presence of different concentrations of glyphosate was tested on Caso-modified agar (3.5 % caso agar, 0.3 % yeast extract, 0.3 % casein hydrolysate, 0.03 % FeSO4 and 0.0.3 % natrium pyruvate, 0.4 % active charcoal and 1 % agar).

Effect of Glyphosate on Sporozoites

Sporulated Eimeria tenella oocysts (isolate LE-01 Eten-05/1) were kindly supplied by Institute of Parasitology, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany. Sporozoites of E. tenella were excysted as described [56]. In brief, after surface sterilization with bleach, the oocyst walls were broken using 0.5-mm glass beads (Biospec product, Bartleville, OK, USA). Thereafter, the sporozoites were recovered from sporocysts by enzymatic excystation using 0.025 trypsin (w/v) (Carl Roth, Karlsruhe, Germany), 10 mM MgCl2 (w/v) and 1 % sodium taurocholic acid (w/v) (Sigma, Taufkirchen, Germany) at 41 °C for 60–90 min. The excysted sporozoites were purified by anion exchange method [48]. The pellet sporozoites were collected carefully from the bottom of the micro-tubes and washed three times with phosphate-buffered saline (PBS). To study the effect of glyphosate on E. tenella, sporozoites were incubated at 37 °C overnight with 1.20, 0.60, 0.30, 0.15 and 0.075 mg glyphosate in 1 ml Dulbecco’s modified Eagle’s medium (DMEM) containing 5 % newborn calve serum. The viability of sporozoites was determined by the trypan blue staining method [56]. In short, sporozoites suspensions were exposed to the same volume of 0.5 % trypan blue in 0.1 M phosphate buffer, pH 7.0. The non-stained parasites were regarded as viable and those stained as dead. Examination of morphological alterations and/or viability was done and images were captured using an inverted microscope (Leica DM IRB, Bensheim, Germany).

Results

Effect of Glyphosate on Potential Pathogens and Beneficial Bacteria

Most of tested pathogenic bacteria were highly resistant to glyphosate; however, most of tested beneficial bacteria were found to be moderate to highly susceptible (Table 2). The herbicide formulated glyphosate at a concentration of 1.2 mg/ml inhibited the growth and BotNT expression (Tables 2, 3). Supplementation of the medium with 1.2 mg/ml glyphosate reduced the cell numbers of C. botulinum type A and B after 5 days of cultivation. C. perfringens, Salmonella Gallinarum, Salmonella Typhimurium, Salmonella Entritidis and E. coli showed to be highly resistance to glyphosate (MIC value 5 mg/ml) (Table 2). L. casei, L. buchneri, L. harbinensis, Staphylococcus aureus and Staphylococcus lentus were moderately resistant to glyphosate (MIC value 0.60 and 0.30 mg/ml, respectively). On the other hand, with the exception of Lactobacillus spp., all tested beneficial bacteria including E. faecalis, E. faecium and B. badius, B. cereus and B. adolescentis were highly sensitive to glyphosate with MIC value of 0.15, 0.15, 0.30 and 0.075 μg/ml, respectively. Colonies morphology showed no significant differences between control and glyphosate-treated bacteria with glyphosate. Microscopically, Lactobacillus spp. B. vulgatus and E. tenella showed swelling and corrugated cell wall.
Table 2
Inhibitory effect of glyphosate on different bacteria
Genus/species
MIC value of glyphosate (mg/ml)a
Bacterial countb
Treated at MIC value (mean ± SD, n = 3)
Untreated (mean ± SD, n = 3)c
Bacillus badius
0.150
2.24 ± 0.49
8.90 ± 0.44
Bacillus cereus
0.300
2.75 ± 0.68
8.08 ± 0.12
Bacteriodes vulgatus
0.600
3.54 ± 0.31
7.37 ± 0.10
Bifidobacterium adolescentis
0.075
3.87 ± 0.50
8.67 ± 0.48
0.150
3.07 ± 0.50
9.00 ± 0.70
Campylobacter jejuni
0.150
3.90 ± 0.50
9.54 ± 0.97
C. perfringens
5.000
3.37 ± 0.89
8.30 ± 0.28
C. botulinum type A
1.200
4.00 ± 0.50
8.16 ± 0.32
C. botulinum type B
1.200
3.56 ± 0.45
7.60 ± 0.57
E. coli
1.200
3.15 ± 0.24
8.00 ± 0.34
E. coli 1917 strain Nissle
1.200
2.35 ± 0.24
7.26 ± 0.21
Enterococcus faecalis
0.150
2.00 ± 0.45
8.49 ± 0.58
Enterococcus faecium
0.150
2.01 ± 0.34
7.06 ± 0.95
Lactobacillus buchneri
0.600
4.00 ± 0.88
8.00 ± 0.22
Lactobacillus casei
0.600
4.74 ± 0.56
8.28 ± 0.35
Lactobacillus harbinensis
0.600
5.30 ± 0.44
8.40 ± 0.32
Riemerella anatipestifer
0.150
4.00 ± 0.50
7.88 ± 0.50
Salmonella Enteritidis
5.000
2.35 ± 0.26
8.28 ± 0.16
Salmonella Gallinarum
5.000
2.15 ± 0.33
8.68 ± 0.20
Salmonella Typhimurium
5.000
2.75 ± 0.68
8.03 ± 0.16
Staphylococcus aureus
0.300
5.74 ± 0.58
9.00 ± 0.10
Staphylococcus haemolyticus
0.300
5.74 ± 0.32
8.08 ± 0.16
Staphylococcus lentus
0.300
3.90 ± 0.44
8.08 ± 0.14
aMinimal inhibitory concentration (MIC) of Glyphosate
bMean of quantitative bacterial counts expressed as reciprocal log10
cBacterial counts without glyphosate treatment (control)
Table 3
Effect of different glyphosate concentrations on C. botulinum types A and B growth and BotNT expression
Glyphosate concentration (mg/ml)
C. botulinum type Aa
C. botulinum type B
CFUb (mean ± SD, n = 3)
BotNT type Ac (mean ± SD, n = 3)
CFU (mean ± SD, n = 3)
BotNT type B (mean ± SD, n = 3)
0
8.16 ± 0.32
2,400 ± 378
7.60 ± 0.57
1,350 ± 270
0.6
6.12 ± 0.67
2,250 ± 126
5.50 ± 0.18
470 ± 60
1.2
4.00 ± 0.50
130 ± 89
3.56 ± 0.45
70 ± 19
2.4
<103
0.0
<103
0.0
5.0
<103
0.0
<103
0.0
a Clostridium botulinum spores (10cfu/ml) in reinforced clostridial medium (RCM) broth incubated with different glyphosate concentrations anaerobically for 5 days at 37 °C
b Clostridium botulinum quantified using the most probable number (MPN) estimation method and expressed as reciprocal log10
cBotulinum neurotoxines measured by ELISA [30]

Effect of Glyphosate on E. tenella Sporozoites

In comparison to untreated control, glyphosate at concentrations of 0.6 mg/ml clearly affected the sporozoites morphology after exposure to glyphosate for 24 h. E. tenella sporozoites showed morphological changes including swelling and corrugated cell wall. At concentration of 0.3 mg/ml, few sporozoites were affected. However, at 0.15 mg/ml, there was no visible effect on sporozoites morphology. At 1.20 mg and 2.4 mg/ml glyphosate, all sporozoites were destroyed.

Discussion

Glyphosate is believed to be the major active principle in the herbicide through its inhibition of EPSPS. It blocks EPSPS, a key enzyme involved in the biosynthesis of aromatic amino acids, naturally present in plants, fungi and some bacteria [49]. The present investigation was carried out to determine the effects of glyphosate on different potential pathogens and beneficial bacteria in the aim to highlight its impact on chicken GIT microbiota. Glyphosate showed different effects according to the tested microorganisms, similar results were described [9, 13, 49]. Most of beneficial poultry microbiota as E. faecalis, E. faecium and B. adolescentis are highly sensitive to glyphosate (Table 2). However, these concentrations did not inhibit growth or BotNT production of C. botulinum type A and B. It is worthy to mention that enterococci are able to suppress growth and toxin production of C. botulinum. The loss of most enterococci from the GIT microbiota may lead to an increase of BotNT production in animals [27]. Special strains of C. perfringens, C. sporogenes and B. cereus were able to antagonize C. botulinum types A, B, F and E [51]. Enterococci were able to inhibit C. botulinum growth [54]. The authors investigated 14 strains of C. botulinum (three type A, four type B, three type E, two type F and two type G), 88 anaerobic and 64 facultative anaerobic isolates using zone inhibition procedures. Strains of bifidobacteria, lactobacilli, propionibacteria and enterococci inhibited the growth of C. botulinum, as did several strains of the Bacteroides fragilis group. C. ramosum was the only member of the genus Clostridium which inhibits C. botulinum type G and F strains. Most of the investigated C. botulinum strains were inhibited in co-cultural experiments with intact faecal samples. Sullivan et al. [54] thought that the inhibition of C. botulinum spores or vegetative cells indicated that the normal flora of healthy individuals had a bacteriostatic, rather than a bactericidal effect on the growth of C. botulinum and concluded that infant botulism may result, in part, from the absence of inhibitory organisms in the normal flora of the infant intestine. Recently, we confirmed that E. faecalis, E. faecium and B. badius inhibited the C. botulinum types A, B, D and E growth and BotNT expression [50]. The loss of such microbiota could be induced by contamination of feeds with residual glyphosate. In vitro toxicity of 0.075–0.30 mg/ml glyphosate inhibited E. faecalis, B. badius and Bifdobacter adolescentis (Table 2). Such concentrations could directly or indirectly disturb the normal gut bacterial community. The maximum residue levels (MRLs) of soya bean, maize, cereal grains, cotton seed, alfalfa, hay, sorghum straw, wheat and wheat straw were agreed by the United Nations Food and Culture Organization’s to be 20, 5.0, 30, 40, 500, 500, 50, 200 and 300 mg/kg [58]. The residue levels of glyphosate in dairy cattle, swine and poultry feeds are 381, 11.5 and 11.4 ppm, respectively [19]. However, data on the real presence of glyphosate and its metabolite AMPA in feed from glyphosate sprayed crops are sparse. Glyphosate residue differs from country to country (in some countries glyphosate is sprayed out of control) and even within a country depending on the quantity and frequency of glyphosate application. Also, the maximum daily intake (MDI) of glyphosate depends on the ration composition and the percent of each component in the ration. Some poultry and cattle feed samples in Germany were found to have 0.4–0.9 mg glyphosate/kg (Data not published). On the other hand, glyphosate were determined in water samples from a transgenic soybean cultivation area located near to tributaries streams of the Pergamino–Arrecifes system in the north of the province of Buenos Aires, Argentina ranged from 0.10 to 0.70 mg/l [43]. Glyphosate daily intake could be hazardous if feed and/or water contain high glyphosate residues; further work is urgently required to determine the real glyphosate residues in animal feed originated from different countries. Also, it is worthy to mention that, glyphosate affects bacterial community indirectly. It has high ability to complex several divalent cationic nutrients such as calcium, magnesium, manganese and iron [1, 12, 32, 37]. These cationic nutrients easily bind to the glyphosate molecule via the carboxyl and phosphonate groups forming poorly soluble or very stable complexes. The maintenance of intracellular homoeostasis of metal ions is crucial for survival of these bacteria. In particular, zinc, manganese and copper are very important metal ions, as they are co-factors of many enzymes, are involved in oxidative stress defence and have a role in the immune system of the host. DNA microarray experiments revealed that these minerals regulate the growth of E. faecalis V583 [14]. In a study performed in our laboratory on a cattle farm infected with C. botulinum, there were a correlation between the presence of enterococci in cow faeces and C. botulinum-associated diseases. Cows that were poorly colonized by enterococci showed in 68 % symptoms of C. botulinum-associated diseases. Also, these cows showed a significant reduction of trace elements in blood. On the other hand, cows with good colonisation by enterococci rarely displayed signs of botulinum diseases (9 %) (Data not published). Thus, the suppression of C. botulinum by enterococci and the exquisite toxicity of glyphosate to enterococci but not C. botulinum observed in this study may be important in the understanding of what is termed chronic botulism in farm animals.
The microbiota of the chicken’s GIT has received increased attention as the focus of efforts to minimise food-borne illness in humans, to improve animal nutrition and to reduce dependence non-therapeutic antibiotic growth promotions [8, 21, 26, 38]. Bifidobacteria are considered beneficial microorganisms and thought to create conditions unfavourable to the growth of pathogens [26]. In the present study, bifidobacteria showed to be highly sensitive to glyphosate which disturb the gut microbiota. On the other hand, Salmonella infections are one of the most important public health concerns worldwide. In general, Salmonella species are widely distributed in the environment and cause a diverse spectrum of diseases in human and animals. Poultry products, eggs and meat are considered to be one of the main sources of human food-borne infections caused by Salmonella [17]. In the present study, Salmonella Enteriditis, Salmonella Typhimurium and Salmonella Gallinarum were highly resistant to glyphosate (MIC value = 5 mg/ml). However, glyphosate may affect on Salmonella indirectly by reducing beneficial microorganisms as bifidobacter which create unfavourable conditions for Salmonella [26]. Also pathogenic E. coli and E. coli 1917 strain Nissle were found also resistant to glyphosate (MIC value = 5 mg/ml). There are several mechanisms by which bacteria and plants are naturally resistant or become resistant to glyphosate: overproduction of EPSPS via gene amplification or increased mRNA biosynthesis, amino acid alterations of EPSPS or natural resistance seen with bacterial class II EPSPS enzymes. This last mechanism of resistance has been utilized for the development of Monsanto’s genetically modified Roundup Ready® crops, which have been successfully transformed with the naturally glyphosate resistant EPSPS from Agrobacterium tumefaciens spp. strain CP4 [10]. It was shown that two single-site amino acid alterations could confer resistance to glyphosate: G96A [18, 53] and P101S [53] (numbering according to E. coli EPSPS). Also, EPSPS from Staphylococcus aureus is insensitive to glyphosate [45]. Glyphosate inhibits the growth of Lactobacillus spp. at 0.6 mg/ml and showed morphological changes including swelling and corrugated cell wall. Clair et al. [13] found that glyphosate inhibited the growth of L. delbrueckii subsp. bulgaricus (food microorganism) at a concentration of 1,000 ppm and also the cell contents leaks out. Antagonism between C. botulinum and bacterial members of the microecosystem are well known [11, 41, 42, 57]. Campylobacters are very important cause of food-borne human diseases. Campylobacteriosis (campylobacter infection) has been described as an emerging food-borne disease [24], and they are now said to be the major cause of bacterial gastroenteritis in humans [31]. Poultry have been reported by several authors to be the leading reservoir for campylobacters, and thus, poultry meat and products are implicated as the leading source of human campylobacteriosis [36]. In the present study, C. coli and C. jejuni were found to be susceptible to glyphosate (MIC value = 0.15 mg/ml). However, it is worthy to mention that some probiotics as L. acidophilus, L. fermentum, Bifidobacterium bifidum and Bifidobacterium longum reduce the pH and create unfavourable conditions for Campylobacter [34]. A reduction of such beneficial bacteria could be also a predisposing factor for campylobacteriosis. In conclusion, glyphosate showed differences in sensitivity between potential pathogens and beneficial microbiota in chicken. A reduction of beneficial bacterial species in the GIT microbiota by ingestion of glyphosate contaminated feed could disturb the normal gut bacterial community. Also, the toxicity of glyphosate to Enterococcus spp. and B. badius could be a significant predisposing factor that is associated with the increase in C. botulinum mediated diseases by suppressing the antagonistic effect of these bacteria on C. botulinum. Investigation on the distribution of glyphosate and its metabolites in poultry feed, their kinetic and the modulatory effect on gut microbiota are in progress. Also, evaluation of genetic mutations induced by glyphosate in different bacteria should be considered.
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