
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39251613
68968
10.1038/s41598-024-68968-6
Article
The effect of combinations of a glyphosate-based herbicide with various clinically used antibiotics on phenotypic traits of Gram-negative species from the ESKAPEE group
Zerrouki Hanane 12
Hamieh Aïcha 12
Hadjadj Linda 12
Rolain Jean-Marc jean-marc.rolain@univ-amu.fr

23
Baron Sophie Alexandra sophie.baron.2@univ-amu.fr

23
1 https://ror.org/035xkbk20 grid.5399.6 0000 0001 2176 4817 MEPHI, Faculté de Médecine et de Pharmacie, Aix Marseille University, 19-21 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
2 https://ror.org/0068ff141 grid.483853.1 0000 0004 0519 5986 IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
3 https://ror.org/035xkbk20 grid.5399.6 0000 0001 2176 4817 APHM, MEPHI, Faculté de Médecine et de Pharmacie, Aix Marseille University, 19-21 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
9 9 2024
9 9 2024
2024
14 2100628 11 2023
30 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The emission of glyphosate and antibiotic residues from human activities threatens the diversity and functioning of the microbial community. This study examines the impact of a glyphosate-based herbicide (GBH) and common antibiotics on Gram-negative bacteria within the ESKAPEE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli). Ten strains, including type and multidrug-resistant strains for each species were analysed and eight antibiotics (cefotaxime, meropenem, aztreonam, ciprofloxacin, gentamicin, tigecycline, sulfamethoxazole-trimethoprim, and colistin) were combined with the GBH. While most combinations yielded additive or indifferent effects in 70 associations, antagonistic effects were observed with ciprofloxacin and gentamicin in five strains. GBH notably decreased the minimum inhibitory concentration of colistin in eight strains and displayed synergistic activity with meropenem against metallo-β-lactamase (MBL)-producing strains. Investigation into the effect of GBH properties on outer membrane permeability involved exposing strains to a combination of this GBH and vancomycin. Results indicated that GBH rendered strains sensitive to vancomycin, which is typically ineffective against Gram-negative bacteria. Furthermore, we examined the impact of GBH in combination with three carbapenem agents on 14 strains exhibiting varying carbapenem-resistance mechanisms to assess its effect on carbapenemase activity. The GBH efficiently inhibited MBL activity, demonstrating similar effects to EDTA (ethylenediaminetetraacetic acid). Chelating effect of GBH may have multifaceted impacts on bacterial cells, potentially by increasing outer membrane permeability and inactivating metalloenzyme activity.

Keywords

Antibiotics
Carbapenem
ESKAPEE group
Glyphosate-based herbicide
Gram-negative bacteria
Subject terms

Bacterial pathogenesis
Antibiotics
issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Glyphosate is a broad-spectrum weed-killing active molecule and the world’s most widely used herbicide1. Its annual use in recent years has been estimated to be between 600 thousand 750 thousand tonnes, with predictions that this could increase to 920 thousand tonnes by 20252. Glyphosate-based herbicide (GBH) is commonly used to control weeds in agricultural fields and urban areas such as industrial sites, public parks, and infrastructures3. Glyphosate and the primary product of its degradation, aminomethylphosphonic acid (AMPA), have been detected in soil, water (including surface water, groundwater, and drinking water)4, foodstuffs5, milk, urine and faeces6. Glyphosate functions as a herbicide by targeting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme, crucial in the shikimate pathway responsible for synthesising aromatic amino acids in plants. This target is also found in microorganisms, imparting additional antimicrobial properties to glyphosate (Patent No.: US 7,771,736, 2010)7,8. Moreover, aside from its antimicrobial attributes, glyphosate, which was initially patented for its chelating properties (Patent No.: US 3,160,632, 1964), can form complexes with various metal ions such as Cu9, Cd10, Zn11, and Mn12 upon contact with soil and water. This impacts the availability of essential metals to plants and microorganisms, potentially altering their metabolic activity13.

Consequently, glyphosate poses a potential threat to microbial biodiversity, as evidenced by previous studies examining the composition of species in the rhizosphere14, zooplankton15, as well as dysbiosis observed in the intestinal microbiota of bees16, earthworms17, and rats18.

Microorganisms are pivotal in preserving biodiversity, undertaking essential physiological and metabolic functions crucial for the integrity of the environment and host organisms19,20.

Moreover, the co-occurrence of other antimicrobial substances exacerbates the health and ecological risks posed by glyphosate21.

Peri-urban regions serve as significant reservoirs of antibiotics, primarily sourced from industrial and municipal waste, along with agricultural activities such as animal feed supplementation, organic fertilisation employing cattle manure, and wastewater irrigation22,23.

The amendment of organic fertilizers and manures to increase the productivity of agriculture is a common practice, but the presence of antibiotic residues potentially boosts antibiotic resistance in agricultural soils24. Several research studies have consistently found antibiotic residues and multidrug-resistant strains in agricultural soils. These include antibiotics from various clinically relevant classes such as tetracyclines, sulphonamides, ciprofloxacin, and carbapenems22,23,25–27.

The use of GBHs and antibiotics together in farming and peri-urban areas poses a serious threat to the environment. Their accumulation can harm the diversity and functions of microbial communities, ultimately impacting agricultural productivity as well as human and animal health28,29. Recently, emphasis has been placed on the link between glyphosate exposure and the emerging antibiotic resistance in enteric and environmental opportunistic bacteria, particularly in bacterioplankton and soil communities30,31, as well as Pseudomonas aeruginosa32, Escherichia coli, and Salmonella spp. strains33–35. Furthermore, it was reported that glyphosate exposure increases cell membrane permeability, which can lead to increased horizontal transfer of multidrug-resistant plasmids36,37. The accumulation of glyphosate and antibiotics is a pressing issue that threatens environmental safety and global health. Despite this, only a few studies have examined their combined impact on opportunistic pathogenic microorganisms, particularly bacteria of the ESKAPEE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterobacter spp., and E. coli). We focused on Gram-negative bacteria (GNB) from this group (A. baumannii, P. aeruginosa, and Enterobacteriaceae) that are highly resistant to several antibiotics and which have been classified as top priority (priority 1: critical) by the WHO, requiring new control agents or emergency measures to manage their spread38. Additionally, these Gram-negative opportunistic pathogen species are omnipresent in soil, water, and human/animal microbiota and are known to cause severe infections and outbreaks, often resulting in high mortality rates39.

Our primary hypothesis is that exposure to GBHs could induce modifications in the antibiotic susceptibility levels of clinically significant bacterial strains, such as GNB in the ESKAPEE group. To investigate this hypothesis further, we examined the impact of the combination of a GBH and commonly used medical and veterinary antibiotics on GNB in the ESKAPEE group, including both susceptible and MDR strains. Subsequently, based on our observations, we investigated the effect of a GBH on outer membrane permeability and metallo-β-lactamase (MBL) activity.

Results

Minimum inhibitory concentration assay

All strains tested in this study and their antibiotic susceptibility profiles are described in Table 1 (details of antibiotics susceptibilities are available in Supplementary Table 1). The minimum inhibitory concentration (MIC) of the GBH was closely distributed among strains within the same species, but there was a clear difference between the various species tested (MIC values ranged from 2 g/L to 10 g/L, corresponding to 1.5 g/L to 7.5 g/L of glyphosate acid) (Supplementary Table S2). Most strains, including both type and MDR strains of K. pneumoniae and E. coli and the E. cloacae type strain, showed growth inhibition at a concentration of 4 g/L to 6 g/L of GBH (corresponding to 3 g/L to 4.5 g/L of glyphosate acid). The highest GBH-MICs (8 g/L to 10 g/L, equivalent to a glyphosate acid concentration of 6 g/L to 7.5 g/L) were observed in both P. aeruginosa strains and the MDR E. cloacae isolate. The lowest GBH-MIC (2 g/L) equivalent to a glyphosate acid concentration of 1.5 g/L was measured in both A. baumannii strains tested.Table 1 Details of strains used in checkerboard assays.

Stains and no	Origin
(year/country)	Resistant antibiotic susceptibility testing phenotype	Used for checkerboard combinations assays	ARGs of interest	Reference	GenBank accession No	
A. baumannii (CIP 70.10)	Type strain	ATM	Preliminary ATBs + GBH;

Carbapenems + GBH

	–	–	–	
A. baumannii (Q9428)	Clinical isolate

(2012/ Iran)

	TIC, TIM, TPZ, ATM, CAZ, FEP, MER, IPM, RA, SXT, AK, CIP, GN	Preliminary ATBs + GBH;

Carbapenems + GBH

	blaOXA-23	Hadjadj et al

201840

	NKXQ00000000	
A. baumannii (Q9426)	Clinical isolate

(2012/Liban)

	TIC, TIM, TPZ, ATM, CAZ, FEP, MER, IPM, SXT, CIP	Carbapenems + GBH	blaNDM*	Rafei et al

201441

	–	
K. pneumoniae (CIP 82.91)	Type strain	AMX	Preliminary ATBs + GBH;

Carbapenems + GBH

	–	–	–	
K. pneumoniae (Q9432)	Clinical isolate

(2008/Israel)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, IPM, FF, NIT, SXT, AK, CIP	Preliminary ATBs + GBH;

Carbapenems + GBH

	blaKPC	Lalaoui et al

201842

	–	
K. pneumoniae (P1571)	Clinical isolate

(2010/France)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, IPM, FF, NIT, SXT, AK, CIP, TET, GN	Carbapenems + GBH	blaNDM*	Diene et al

201243

	HQ328085	
K. pneumoniae (Q9430)	Clinical isolate

(2018/France)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, SXT, AK, CIP, TET, GN	Carbapenems + GBH	blaOXA-48	–	–	
K. pneumoniae (Q9427)	Clinical isolate

(2012/France)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, NIT, SXT, AK, CIP, TET, GN	Carbapenems + GBH	blaVIM*	–	–	
K. pneumoniae (Q9429)	Clinical isolate

(2018/France)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, IPM, FF, NIT, SXT, CIP	Carbapenems + GBH	Membrane impermeability	–	–	
E. coli

(CIP 7624)

	Type strain	–	Preliminary ATBs + GBH; Carbapenems + GBH	–	–	–	
E. coli

(Q1950)

	Clinical isolate

(2019/ Lebanon)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, IPM, SXT, AK, CIP, TET, GN	Preliminary ATBs + GBH; Carbapenems + GBH	blaNDM*	Chamieh et al

202144

	JAFEVK010000000	
E. coli

(P1570)

	Poultry isolate

(2013/ Lebanon)

	AMX, AMC, TPZ, ETP, SXT	Carbapenems + GBH	blaOXA-48	Al Bayssari et al

201445

	KM390023	
E. coli

(Q9431)

	Clinical isolate

(2011/France)

	AMX, AMC, MEC, NIT, TET	Carbapenems + GBH	blaGES	–	–	
P. aeruginosa (CIP 76.110)	Type strain	RA, SXT, MIN	Preliminary ATBs + GBH; Carbapenems + GBH	–	–	–	
P. aeruginosa (Q5596)	Clinical isolate

(2016/Pakistan)

	TIC, TIM, TPZ, ATM, CAZ, FEP, MER, IPM, RA, SXT, AK, CIP, MIN, GN	Preliminary ATBs + GBH;

Carbapenems + GBH

	blaVIM*	Hadjadj et al

202146

	–	
P. aeruginosa (Q9424)	Clinical isolate

(2016/Pakistan)

	TIC, TIM, TPZ, ATM, CAZ, FEP, MER, IPM, RA, SXT, AK, CIP, MIN, GN	Carbapenems + GBH	Alterations of porin OprD	Hadjadj et al

202146

	–	
E. cloacae

(CIP 104,674)

	Type strain	AMX, AMC, NIT, SXT	Preliminary ATBs + GBH;

Carbapenems + GBH

	–	–	–	
E. cloacae (P7536)	Clinical isolate

(2017/France)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, NIT, SXT, AK, CIP, TET, GN	Preliminary ATBs + GBH;

Carbapenems + GBH

	blaOXA-48	Hadjadj et al

202247

	CP071788	
E. cloacae (Q9425)	Clinical isolate

(2016/Pakistan)

	AMX, AMC, FEP, TPZ, MEC, CRO, ETP, SXT, GN	Carbapenems + GBH	blaNDM*	Hadjadj et al

202146

	-	
ARGs: antibiotic resistance genes, * Class B carbapenemase (MBL).

Antibiotics tested on non-fermenting Gram-negative bacilli strain: TIC: Ticarcillin, TIM: Ticarcillin-clavulanic acid, TPZ: Piperacillin-tazobactam, ATM: Aztreonam, CAZ: Ceftazidime, FEP: Cefepime, MER: Meropenem, IPM: Imipenem, FF: Fosfomycin, RA: Rifampicin, SXT: Sulfamethoxazole-trimethoprim, AK: Amikacin, CIP: Ciprofloxacin, MIN: Minocycline, CS: Colistin, GN: Gentamicin.

Antibiotics tested on enterobacteria strains: AMX: Amoxicillin, AMC: Amoxicillin-clavulanic acid, FEP: Cefepime, TPZ: Piperacillin-tazobactam, MEC: Mecillinam, CRO: Ceftriaxone, ETP: Ertapenem, IPM: Imipenem, FF: Fosfomycin, NIT: Nitrofurantoin, SXT: Sulfamethoxazole-trimethoprim, AK: Amikacin, CIP: Ciprofloxacin, TET: Tetracycline, CS: Colistin, GN: Gentamicin.

The MDR isolates showed higher MIC values for most antibiotics, except for colistin. Tigecycline was effective on all strains except for P. aeruginosa. It is worth mentioning that E. cloacae-blaOXA-48 (Q9425) showed a low MIC value of 1 mg/L for meropenem, while gentamicin was effective against K. pneumoniae-blaKPC (Q9432) with an MIC of 0.25 mg/L (Supplementary Table S2).

The effect of the GBH in combination with clinically relevant antibiotics

The ten strains were tested with eight combinations of the GBH and antibiotics. Most associations showed either an additive (n = 35) effect or an indifferent effect (n = 35), with only five combinations showing antagonistic or synergistic effects (Table 2). The GBH had an antagonistic effect with ciprofloxacin and gentamicin on the MDR strain of A. baumannii, and only with ciprofloxacin on both the tested strains of E. coli. The strongest antagonistic effect was observed with E. coli, where the MIC of ciprofloxacin increased from 0.003 mg/L to 0.03 mg/L. However, the GBH-colistin combinations had a synergistic effect against the A. baumannii strains tested, with a decrease in the MIC of colistin. For other strains, the effect was either indifferent or additive, but the addition of the GBH reduced the MIC of colistin at least three-fold. For example, the MIC of colistin for E. coli (CIP 7624) decreased from 1 mg/L to 0.06 mg/L, while for E. coli-blaNDM (Q1950) and P. aeruginosa (CIP 76.110), it decreased from 1 mg/L to 0.125 mg/L and from 1 mg/L to 0.01 mg/L, respectively (Table S2 in the supplementary file). Similarly, the colistin MIC of P. aeruginosa-blaVIM (Q5596) decreased from 2 mg/L to 0.007 mg/L, while for E. cloacae (CIP 104,674) and E. cloacae-blaOXA-48 (P7536), the colistin MIC decreased from 2 mg/L to 0.25 mg/L and from 1 mg/L to 0.001 mg/L, respectively (Table S2 in supplementary file). The GBH-meropenem combinations also showed a synergistic effect on MBL producing strains. The addition of the GBH reduced the meropenem MICs from 16 mg/L to 0.06 mg/L for E. coli-blaNDM (Q1950), with a FICI of 0.3, and from 32 mg/L to 0.5 mg/L for P. aeruginosa-blaVIM (Q5596), with a FICI of 0.2 (Table 2).Table 2 FICI values and outcome of a GBH in combination with antibiotics in the checkerboard assay on Gram-negative bacteria of ESKAPEE group. FICIs were calculated based on the concentration of glyphosate-isopropylamine salt in the tested GBH and its equivalence to glyphosate acid. Since the results were similar, only the data for the GBH are reported here.

Antibiotics + GBH
combinations	Cefotaxime
 + 
GBH	Meropenem
 + 
GBH	Aztreonam
 + 
GBH	Tigecycline
 + 
GBH	SXT
 + 
GBH	Gentamicin
 + 
GBH	Ciprofloxacin
 + 
GBH	Colistin
 + 
GBH	
A. baumannii

(CIP 70.10)

	Indifference

(FICI = 1)

	Additive

(FICI = 0.75)

	Indifference

(FICI = 1)

	Additive

(FICI = 0.75)

	Indifference

(FICI = 1)

	Indifference

(FICI = 1.12)

	Indifference

(FICI = 1.24)

	Synergy

(FICI = 0.37)

	
A. baumannii (Q9428)	Indifference

(FICI = 1.25)

	Additive

(FICI = 0.75)

	Indifference (FICI = 1.03)	Indifference

(FICI = 1.25)

	Indifference

(FICI = 1.5)

	Antagonism

(FICI = 2)

	Antagonism

(FICI = 2)

	Synergy

(FICI = 0.37)

	
K. pneumoniae (CIP 82.91)	Indifference

(FICI = 1.16)

	Indifference

(FICI = 1.36)

	Additive

(FICI = 0.9)

	Additive

(FICI = 0.91)

	Additive

(FICI = 0.91)

	Antagonism

(FICI = 2.66)

	Indifference

(FICI = 1.66)

	Additive

(FICI = 0.91)

	
K. pneumoniae (Q9432)	Additive

(FICI = 0.67)

	Additive

(FICI = 0.75)

	Additive

(FICI = 0.72)

	Additive

(FICI = 0.91)

	Additive

(FICI = 0.91)

	Indifference

(FICI = 1.66)

	Indifference

(FICI = 1.66)

	Indifference

(FICI = 1.16)

	
E. coli

(CIP 7624)

	Additive

(FICI = 0.99)

	Indifference

(FICI = 1.17)

	Additive

(FICI = 0.87)

	Additive

(FICI = 0.62)

	Indifference

(FICI = 1)

	Indifference

(FICI = 1.25)

	Antagonism

(FICI = 10.75)

	Additive

(FICI = 0.81)

	
E. coli*

(Q1950)

	Additive

(FICI = 0.62)

	Synergy

(FICI = 0.3)

	Additive

(FICI = 0.75)

	Indifference

(FICI = 1)

	Indifference

(FICI = 1)

	Indifference

(FICI = 1.16)

	Antagonism

(FICI = 2.5)

	Additive

(FICI = 0.62)

	
P. aeruginosa

(CIP 76.110)

	Indifference

(FICI = 1.25)

	Indifference

(FICI = 1.12)

	Indifference

(FICI = 1.25)

	Indifference

(FICI = 1.12)

	Indifference

(FICI = 1.31)

	Indifference

(FICI = 1.24)

	Indifference

(FICI = 1.04)

	Indifference

(FICI = 1.01)

	
P. aeruginosa*

(Q5596)

	Synergy

(FICI = 0.46)

	Synergy

(FICI = 0.21)

	Indifference

(FICI = 1.3)

	Indifference

(FICI = 1.3)

	Additive

(FICI = 0.86)

	Indifference

(FICI = 1.3)

	Indifference

(FICI = 1.8)

	Additive

(FICI = 0.8)

	
E. cloacae

(CIP 104,674)

	Additive

(FICI = 0.75)

	Indifference (FICI = 1.1)	Additive

(FICI = 0.56)

	Additive

(FICI = 0.69)

	Additive

(FICI = 0.67)

	Additive

(FICI = 0.78)

	Additive

(FICI = 0.92)

	Additive

(FICI = 0.78)

	
E. cloacae

(P7536)

	Indifference

(FICI = 1)

	Additive

(FICI = 0.75)

	Additive

(FICI = 0.75)

	Additive

(FICI = 0.75)

	Additive

(FICI = 0.75)

	Additive

(FICI = 0.65)

	Additive

(FICI = 0.65)

	Additive

(FICI = 0.75)

	
Synergistic results are highlighted in bold.

Outer membrane destabilisation of GNBs by the GBH

In combination with the GBH, colistin MICs were reduced by a factor of at least three for eight of the ten strains tested. To investigate further, we assessed the permeabilising effect of GBH by evaluating its ability to render vancomycin active on GNB. Vancomycin is a glycopeptide antibiotic that acts on Gram-positive bacteria but cannot penetrate the outer membrane of GNBs due to its high molecular weight. The combination of a GBH and vancomycin on ten isolates increased bacterial growth inhibition from an average of 9% (2% to 19%) to an average of 97% (94% to 99%) (Fig. 1). These observations suggest that GBH increases outer membrane permeability, facilitating the action of antibiotics such as vancomycin and colistin.Figure 1 The percentage of bacterial growth inhibition in the presence of vancomycin alone, GBH tested alone, and a combination of vancomycin and the tested GBH. The concentration of vancomycin tested was 150 mg/L. The concentration of GBH tested was 0.1 mM (28 mg/L of glyphosate-isopropylamine salt in the tested GBH equivalent to 0.021 g/L of glyphosate acid). *Strain producing class B carbapenemase (MBL).

Investigation of GBH-carbapenem combinations

Our research uncovered significant synergistic effects of the combination of GBH and meropenem on MBL-producing strains. To delve deeper into these findings, we examined the effects of GBH combinations with three distinct carbapenem antibiotics on 19 strains including the five carbapenem-susceptible type strains (K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp. and E. coli), and 14 strains exhibiting varying carbapenem resistance mechanisms (blaKPC, blaGES, blaNDM, blaVIM, blaOXA-23, blaOXA-48, membrane permeability mutations, and porin-deficient OprD). The MIC values of a GBH and carbapenems for all tested strains are shown in Table S3 in the supplementary file.

All six of the MBL-producing strains we tested, namely K. pneumoniae-blaNDM (P1571), K. pneumoniae-blaVIM (Q9427), A. baumannii-blaNDM (Q9426), P. aeruginosa-blaVIM (Q5596), E. cloacae-blaNDM (Q9425), and E. coli-blaNDM (Q1950), showed synergistic effects when combined with the GBH and all three carbapenem agents (Table 3). However, only additive or indifferent effects were found with other isolates, including serine-carbapenemase-producing strains, oxacillinase-positive strains, porin-deficient OprD P. aeruginosa, K. pneumoniae with membrane impermeability, and type strains (Table 3).Table 3 FICI values and outcome of a GBH in combination with carbapenems in the checkerboard assay on carbapenem-resistant strains. FICIs were calculated based on the concentration of glyphosate-isopropylamine salt in the tested GBH and its equivalence to glyphosate acid. Since the results were the same, only the data for the GBH are reported here.

Tested strains	Imipenem + GBH	Ertapenem + GBH	Meropenem + GBH	
A. baumannii (CIP 70.10)	Additive (FICI = 0.74)	Indifference (FICI = 1.42)	Additive (FICI = 0.75)	
A. baumannii (Q9428)	Additive (FICI = 0.75)	Additive (FICI = 0.62)	Additive (FICI = 0.75)	
A. baumannii* (Q9426)	Synergy (FICI = 0.31)	Synergy (FICI = 0.26)	Synergy (FICI = 0.28)	
K. pneumoniae (CIP 82.91)	Additive (FICI = 0.83)	Indifference (FICI = 1.66)	Indifference (FICI = 1.36)	
K. pneumoniae (Q9432)	Indifference (FICI = 1.16)	Additive (FICI = 0.78)	Additive (FICI = 0.75)	
K. pneumoniae* (P1571)	Synergy (FICI = 0.37)	Synergy (FICI = 0.26)	Synergy (FICI = 0.26)	
K. pneumoniae (Q9430)	Additive (FICI = 0.85)	Additive (FICI = 0.85)	Indifference (FICI = 1.1)	
K. pneumoniae* (Q9427)	Synergy (FICI = 0.43)	Synergy (FICI = 0.40)	Synergy (FICI = 0.42)	
K. pneumoniae (Q9429)	Additive (FICI = 0.8)	Additive (FICI = 0.8)	Additive (FICI = 0.8)	
E. coli (CIP 7624)	Additive (FICI = 0.58)	Additive (FICI = 0.92)	Indifference (FICI = 1.17)	
E. coli* (Q1950)	Synergy (FICI = 0.36)	Synergy (FICI = 0.3)	Synergy (FICI = 0.3)	
E. coli (P1570)	Additive (FICI = 0.74)	Additive (FICI = 0.73)	Indifference (FICI = 1.23)	
E. coli (Q9431)	Additive (FICI = 0.75)	Indifference (FICI = 1.2)	Additive (FICI = 0.92)	
P. aeruginosa (CIP 76.110)	Indifference (FICI = 1)	Additive (FICI = 0.81)	Indifference (FICI = 1.12)	
P. aeruginosa* (Q5596)	Synergy (FICI = 0.43)	Synergy (FICI = 0.41)	Synergy (FICI = 0.21)	
P. aeruginosa (Q9424)	Indifference (FICI = 1.16)	Additive (FICI = 0.91)	Additive (FICI = 0.91)	
E. cloacae (CIP 104,674)	Indifference (FICI = 1.1)	Indifference (FICI = 1.1)	Indifference (FICI = 1.1)	
E. cloacae (P7536)	Additive (FICI = 0.75)	Additive (FICI = 0.62)	Additive (FICI = 0.75)	
E. cloacae* (Q9425)	Synergy (FICI = 0.46)	Synergy (FICI = 0.41)	Synergy (FICI = 0.41)	
*: Strain producing class B carbapenemase (MBL).

Synergistic results are highlighted in bold.

Study of the effect of GBH on MBL-producing strains

The test aims to differentiate MBL-producing strains from other carbapenemases by studying the effect of EDTA on their enzymatic activity. EDTA inactivates metalloenzymes by binding to Zn2+ ions. We reversed the experiment to assess the chelating effect of a GBH, testing a collection of strains with known resistance mechanisms. The addition of GBH to bacterial suspensions produced an effect like that of EDTA for all strains tested (Fig. 2). An inhibition zone was observed around meropenem discs supplemented with GBH and EDTA for the six MBL-producing enterobacteria. In contrast, we observed no inhibition zone around the meropenem discs which were not supplemented (Fig. 3). Following the addition of ZnSO4 in combination with GBH, we observed a restoration of MBL activity. The same result was obtained with a mixture of ZnSO4 and EDTA.Figure 2 A heat map was generated to illustrate the degradation or non-degradation of carbapenems, determined by employing the modified carbapenem inactivation method combined with the addition of ZnSO4 (zCIM), EDTA (eCIM), GBH (gCIM), ZnSO4-EDTA, and ZnSO4-GBH. This approach aimed to evaluate the influence of the tested GBH on strains producing MBLs. *Strain producing class B carbapenemase (MBL). MP mutation: membrane permeability mutation.

Figure 3 Photo of mCIM, eCIM, and gCIM test results. We present the results obtained on K. pneumoniae strains, including one antibiotic-sensitive type strains and three carbapenemase-producing strains of classes A, B, and D. T- is the name of the tube containing only TSB, while the symbols EDTA and Gly have been given to the other tubes supplemented with EDTA (eCIM) and the GBH (gCIM), respectively.

We observed no effect of either EDTA or GBH on type strains, and on strains that do not present metallo-beta-lactamases, OprD-deficient P. aeruginosa, K. pneumoniae with membranous impermeability, class A carbapenemase-producing (blaKPC, and blaGES), and class D carbapenemase-producing (blaOXA-48, blaOXA-23) strains (Fig. 2). Based on these results, chelating properties of glyphosate are presumed as indicated by previous studies48–50.

Discussion

The objective of our work was to study the impact of the combination of a GBH and antibiotics on GNB in the ESKAPEE group. In this study, we observed various effects of this GBH on combinations of bacteria/antibiotics. We found that the addition of the GBH notably alters phenotypic antibiotic sensitivity. At first, our research revealed a specific synergistic effect between GBH and carbapenems on the growth of MBL-producing strains. Unlike other carbapenemase classes (classes A, C, and D), which use a serine-linked acyl intermediate to catalyse β-lactam hydrolysis, class B MBLs require zinc for their activity51, which can be neutralised by metal chelators such as EDTA52. Research suggests that glyphosate's isopropylamine salt has EDTA-like chelating properties, allowing it to form stable complexes with divalent and trivalent metal ions and micronutrients like zinc50. Glyphosate acid acts as a tridentate zwitterionic chelating agent, possessing three functional groups: carboxylate, amine, and phosphonate, with both positive and negative charges53. Additionally, isopropylamine salt is also a chelator, thereby enhancing the chelating potential of the herbicide based on glyphosate isopropylamine salt54.

We observed the reactivation of MBLs after the addition of ZnSO4. Consequently, we conclude that the GBH is capable of sequestering metal ions in its environment and has a potential impact on various enzymatic activities reliant on zinc as a cofactor, including MBL activity51,55. Although this may seem like a positive development in the fight against MDR strains, it can seriously affect the health of plants, animals, and humans. The MBL superfamily is widespread and has been described in all life forms, including prokaryotic organisms such as bacteria and archaea, as well as eukaryotes, including humans56. This class of enzymes is heterogeneous and has been implicated in several physiological processes, such as the transport of molecules across membranes, communication through quorum sensing, and the hydrolysis of antibiotics (β-lactamase), anticancer drugs (cisplatin and mitomycin C), nucleic acids, ascorbic acid and xenobiotics (e.g. glyoxalase and lactonase enzymes)57. We have shown the effect of the GBH on beta-lactamases, but it would be interesting to evaluate its effect on other MBL-fold enzymes.

In our work, we also highlighted an effect on the outer membrane that could be linked to the chelating properties of GBH. The GBH addition rendered our strains susceptible to colistin and vancomycin. Vancomycin is naturally active only against Gram-positive bacteria. However, GNBs become susceptible to this antibiotic when the structure of their outer membrane is disrupted58. Studies have shown that adding chelating agents such as EDTA can potentiate the effect of colistin on MDR strains such as Salmonella spp59. and K. pneumoniae60, leading to a decrease in their MICs to colistin and can make vancomycin effective against E. coli MDR strains61,62. These results align with our observations. We suggest that the chelating properties of GBH, which may sequester divalent cations Mg2+ or Ca2+, thereby increase the permeability of the outer membrane and facilitate the entry of antibiotics63. Additionally, we suggest that the chelating effect of glyphosate-isopropylamine saltcould have multifaceted impacts on bacterial cells, potentially rendering the outer membrane more permeable and inactivating metalloenzymes.

In our study, we found that the association of GBH and gentamicin had antagonistic effects on the A. baumannii-blaOXA-23 (Q9428) strain and the K. pneumoniae (CIP 82.91) strain. Similarly, the combination of GBH and ciprofloxacin had an antagonistic reaction on the A. baumannii-blaOXA-23 (Q9428) strain and both E. coli strains tested. Other studies have also shown that being exposed to glyphosate increases the MICs of ciprofloxacin and aminoglycosides in E. coli and Salmonella enterica serovar Typhimurium strains34,35. The authors suggest that this could be an adaptive response leading to a cross-response to antibiotics by increasing membrane efflux31. However, it has been demonstrated that the targeted deletion of efflux pump genes may counteract the increased tolerance to kanamycin and ciprofloxacin in E. coli and S. enterica serotype Typhimurium, when exposed to GBH35.

The main limitation of our study lies in the concentrations of GBH used in the preliminary tests. Our experimental approach aimed to explore the effects of combining a GBH and antibiotics through checkerboard tests, using separately determined MICs for each substance64. The GBH exhibited high MICs for our bacterial panel, however, subsequent experiments showed a comparable effect at lower GBH concentrations, particularly with the GBH-supplemented carbapenem inactivation method (gCIM) and the GBH/vancomycin combination tests. The concentration of GBH tested in these experiments was 0.1 mM, (28 mg/L equivalent to 0.021 g/L of glyphosate acid), falling within the acceptable limits of regulated glyphosate for short-term freshwater exposure in the United States (49.9 mg/L) and Canada (27 mg/L)31. It is worth mentioning that we evaluated a GBH formulation containing quaternary ammonium compounds alongside glyphosate isopropylamine salt. We opted for a GBH with quaternary ammoniums due to their reduced toxicity and widespread use as alternatives to GBHs containing polyethoxylated amines (POEAs). While regulatory authorities consider glyphosate as the active ingredient in GBHs, with other chemical compounds presumed to be inert65. We recognise that quaternary ammoniums also possess antibiotic properties, potentially heightening the risks associated with microbial exposure to GBH. In our study, we did not test glyphosate salt alone; rather, we analysed it as part of the complete herbicide formulation, simulating environmental contamination. The impact of the chelating effect of glyphosate-isopropylamine salt has already been demonstrated by altering plant66,67 and algae54 growth and vegetal enzymes50. This chelating activity could varies depending to the tested formulation50, as it has been demonstrated for the combination effect of different GBHs formulation and imipenem on P. aeruginosa32.

The simultaneous presence of herbicides and antibiotics is frequent in agricultural, urban and aquatic environments receiving effluents or leachates from agroecosystems, livestock farms, and clinical settings35,68. The relationship between GBH and antibiotics, and how they impact opportunistic pathogenic and MDR Gram-negative bacteria has been studied only sporadically, even though these bacteria are widespread in areas of human activity and are frequently linked to severe infections. The presence of a pesticide in diverse environments (such as soil, water, and wastewater), where antibiotic concentrations are typically low, may elevate these levels to a degree that inhibits sensitive species and selects tolerant strains35.

These findings raise concerns about the potential risks that GBHs could cause to microbial diversity and global health. In addition to its antimicrobial properties, the chelation capacity of glyphosate, even at low concentrations, enables it to bind to macro- and micronutrients, inhibitors or activators of numerous critical physiological processes in plants, mammals, and microorganisms69. Further investigation is crucial to understand the effects of other glyphosate formulations on microbial antibiotics sensitivity.

Materials and methods

Collection and susceptibility of bacterial strains

We analysed Gram-negative ESKAPEE pathogenic strains, encompassing K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp., and E. coli. For preliminary combination assays of ATBs with the GBH, we selected ten strains (Table 1), including five type strains and five MDR strains: K. pneumoniae (CIP 82.91 and Q9432), A. baumannii (CIP 70.10 and Q9428) P. aeruginosa (CIP 76.110 and Q5596), Enterobacter spp. (CIP 104,674 and P7536), and E. coli (CIP 7624 and Q1950). The type strains were obtained from the Institut Pasteur collection (Paris, France), while the MDR strains were isolated and characterised at the Institut Hospitalier Universitaire Méditerranée Infection (IHU-MI) (Marseille, France). We classified MDR strains according to the definition established by Magiorakos et al., indicating acquired non-susceptibility to at least one agent from three or more classes of antibiotics70. In a subsequent step, to explore the impact of carbapenems and GBH combinations, we expanded our strain collection to include nine carbapenem-resistant strains, each exhibiting various mechanisms of resistance (Table 1). Strain identification was performed using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany), as described previously71. Antibiotic susceptibility of the growing bacteria was determined using the disk diffusion method following guidelines from the European Committee for Antimicrobial Susceptibility Testing (EUCAST, 2022). Two different panels of 16 antibiotics were tested on enterobacteria and non-fermenting GNB (see Table 1 legend). Standard qPCR and PCR assays were conducted to confirm the presence of carbapenemase genes (blaNDM, blaVIM, blaKPC, blaOXA-23, blaOXA-24, blaOXA-48, and blaOXA-58)72.

Chemicals and stock solutions used

We used cation adjusted Mueller–Hinton broth media (CAMHB, Merck KGaA, Darmstadt, Germany) and Trypticase soy agar (TSA, bioMérieux, Marcy-l’Étoile, France) medium for bacterial culture. We chose to investigate antibiotics used in human and veterinary medicine, notably ciprofloxacin, gentamicin, tigecycline, sulfamethoxazole + trimethoprim, and colistin (Sigma Aldrich, Saint-Quentin-Fallavier, France). Additionally, we tested antibiotics exclusively used in human medicine, such as cefotaxime, meropenem, imipenem, ertapenem and aztreonam (Sigma Aldrich, Saint-Quentin-Fallavier, France). For each antibiotic, we prepared a 1 g/L stock solution in deionised water using either a lyophilised or solution formulation. Additionally, we used a commercial GBH (Radikal, Fitogal, Spain), which is a soluble concentrated solution formulated with 360 g/L of glyphosate-isopropylamine salt, corresponding to 270 g/L of glyphosate acid. This formulation also includes a quaternary ammonium as a coformulant, but the specific type and concentration are not disclosed by the manufacturer (Expiration date of approval: 30/04/2028). This solution was diluted to a concentration of 20 mg/mL in a sterile CAMHB medium.

Minimum inhibitory concentration assays

MICs for antibiotics and the GBH were determined using the broth microdilution method. The sterile CAMHB medium was used as a negative control, while the E. coli type strain, with known antibiotic sensibility, served as a positive control. The final concentration of GBH solution in the microtiter plates ranged from 0.0625 g/L to 32 g/L for all strains. Antibiotic concentrations tested ranged from 0.5 mg/L to 32 mg/L for susceptible strains, and from 0.5 mg/L to 256 mg/L for MDR strains. Antibiotic sensitivity was determined using 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich®, St Louis, Missouri, USA). A 0.1% (w/v) TTC solution was prepared and added after 22 h of incubation at 37 °C, followed by an additional two hours of incubation, to determine the MIC73. The experiment was repeated in triplicates and performed independently.

Checkerboard assay

The antibacterial effects of the combination between GBH and the tested antibiotic were assessed using the checkerboard test, as previously described by Sopirala et al.74. Serial dilutions of each antibiotic and a GBH at decreasing concentrations were prepared in CAMHB ranging from 4 × MIC to 1/128 × MIC. Each checkerboard well was streaked with 45 μL of each agent and 10 μL of a bacterial suspension at 105 CFU/mL. Additional rows were used to determine the MIC of each antimicrobial compound alone by adding 90 µL of each agent. For each drug combination, the Fractional Inhibitory Concentration (FIC) and the Fractional Inhibitory Concentration Index (FICI) were calculated according to the equation FICI = FICA + FICG = (MIC of the tested antibiotic in combination with the GBH/MIC of antibiotic alone) + (MIC of GBH in combination with antibiotic /MIC of GBH alone). The interaction was defined as synergy (FICI ≤ 0.5), additive (FICI > 0.5 and < 1), indifferent (FICI > 1 and < 2), and antagonistic (FICI > 2)75,76.

Screening of a GBH effect on the outer membrane

We conducted an experiment to study the impact of GBH on enhancing the activity of colistin and its effect on the stability of the outer membrane of GNB. To do so, we tested the sensitivity of ten strains, including five type strains and five MDRs (used in the preliminary tests and previously cited), to the combination of GBH and vancomycin, which is typically only active against Gram-positive bacteria. We followed the methodology described by Muheim et al., with a few modifications77. We prepared overnight bacterial cultures and diluted them to an optical density of OD600 = 0.01. Subsequently, we added either 150 mg/L of vancomycin, 0.1 mM of the GBH (28 mg/L of glyphosate-isopropylamine salt in the tested GBH equivalent to 21 mg/L of glyphosate acid), or a combination of both vancomycin and GBH (150 mg/L and 0.1 mM, respectively). Next, 100 µL of each mixture was added to the wells of microtiter plates. These plates were then incubated for five hours at 37 °C without shaking, and the optical density at OD600 was measured using a spectrophotometer (Multiskan Spectrum, ThermoFisher Scientific, Waltham, MA, USA). Three growth controls were included: bacterial suspension alone, bacterial suspension with 150 mg/L of vancomycin, and bacterial suspension with 0.1 mM a GBH. A sterility control consisting of sterile growth medium was also included. The bacterial growth inhibition rate was determined using the following equation, which correlates with the absorbance (A) measured in the wells: bacterial growth inhibition (%) = [1 − (Asample − Anegative control / Apositive control − Anegative control)] × 100.

Investigation of the association of the GBH and carbapenem

After the initial results, we aimed to investigate how GBH and carbapenem combinations affect carbapenem-resistant strains. We selected 19 Gram-negative strains from the ESKAPEE group, which included the five type strains mentioned above, and 14 strains exhibiting varying carbapenem resistance mechanisms (A. baumannii-blaOXA-23 (Q9428), A. baumannii-blaNDM (Q9426), K. pneumoniae-blaOXA-48 (Q9430), K. pneumoniae-blaNDM (P1571), K. pneumoniae- blaVIM (Q9427), K. pneumoniae-blaKPC (Q9432), E. coli-blaOXA-48 (P1570), E. coli-blaNDM (Q1950), E. coli-blaGES (Q9431), P. aeruginosa- blaVIM (Q5596), E. cloacae-blaOXA-48 (P7536), E. cloacae-blaNDM (Q9425), OprD-deficient P. aeruginosa (Q9424), and one with membrane permeability mutations, K. pneumoniae (Q9429) (Table 1). We tested three different carbapenem antibiotics (imipenem, ertapenem, and meropenem) and the GBH, individually and in combination, using the experimental procedure reported above.

Study of the effect of a GBH on MBL-producing strains

To explore the potential synergistic impact of carbapenem agents and GBH on MBL strains, the modified carbapenem inactivation method combined with the EDTA-carbapenem inactivation method (eCIM) developed by the Clinical and Laboratory Standards Institute (CLSI) was used, with a few modifications. The EDTA was used in this assay to differentiate MBL from other carbapenemases, specifically class A and D. Additionally, we tested GBH to confirm whether it exhibits similar ion chelating properties. Fourteen strains which were resistant to carbapenems by various means were tested (Table 1). The test was conducted following the description published by Sfeir et al.78. Briefly, a colony of the tested strain was resuspended in three tubes with 2 mL of tryptic soy broth (TSB, bioMérieux) from the overnight culture. The first tube contained only TSB, while the second tube was supplemented with EDTA (ThermoFisher Scientific, Waltham, Massachusetts, USA) to a final concentration of 0.1 mM and used as a positive control to validate the experiment. The third tube had an additional 0.1 mM of GBH. A meropenem disc (10 µL) was placed in all tubes. After four hours of incubation at 35 °C ± 15 min, the discs were placed on Mueller–Hinton (BD) agar plates inoculated with a 0.5 McFarland suspension of a carbapenem-susceptible type strain (E. coli CIP 7624). In a second step, we experimented to determine whether the GBH binds to zinc ions in the active site of MBL enzymes or chelates free zinc ions. To do so, we combined a GBH with the zinc-supplemented carbapenem inactivation method (zCIM)79. We added a meropenem disc to a bacterial suspension grown in TSB supplemented with ZnSO4 (final concentration 1.5 mM) and the GBH (final concentration 0.1 mM). We prepared a bacterial suspension containing ZnSO4 alone (final concentration 1.5 mM) and the GBH alone.

We used previously established thresholds: an inhibition zone ≤ 15 mm indicates carbapenem degradation, while an inhibition zone > 15 mm indicates the absence of carbapenem degradation to interpret the results. We used type strains that are incapable of degrading meropenem, along with strains producing class A and D carbapenemases that would not be affected by EDTA, as negative controls. The addition of EDTA, a known agent for chelating MBLs, served as a positive control in our experiment.

Supplementary Information

Supplementary Tables.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-68968-6.

Author contributions

L.H., J.M.R. and S.A.B.: designed and conceived the study. H.Z., A.H., L.H.: performed the experiments and interpreted the data. H.Z., A.H., L.H. and S.A.B. drafted the manuscript. H.Z., L.H. J.M.R., and S.A.B.: made critical corrections and revisions.  All authors read and approved the final version of the manuscript.

Funding

This work was supported by the French government under the Investissements d'avenir programme managed by the Agence Nationale de la Recherche (ANR), Méditerranée Infection (10-IAHU-03).

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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