Antimicrobial and other beneficial properties of fodder species with potential use in controlling diarrhoeagenic Escherichia coli in cattle

Abstract

Shiga toxin-producing E. coli (STEC) is a diarrhoeagenic pathogen commonly transmitted from cattle to other animals via contaminated meat, milk and environmental water sources, causing a serious public health burden with major economic implications in livestock and human health. Diarrhoeagenic E. coli harbour biofilm-forming and quorum sensing characteristics involved in multidrug resistance (MDR). Multidrug resistance is challenging in combating infectious veterinary and zoonotic diseases, prompting restrictions on prophylactic use of antimicrobials as feed additives, especially for growth promotion in young animals. In addition, microbial infections also contribute to inflammatory and oxidative disorders in immune-compromised young or old animals, negatively affecting production and health. Thus, there is an urgent need to discover novel alternative or complementary feed additives with nutritional value, antimicrobial, antibiofilm, and anti-inflammatory properties to maintain cattle gastrointestinal health, increase growth rate, and maximise production efficiency. In African countries including those in southern Africa, there is substantial application of various plant species in cattle production for use as alternative animal feed or as anti-diarrhoeal remedies against infectious pathogens. Many of the same plant species have been traditionally used to treat various diarrhoeal and inflammatory conditions in humans. Hence, sixteen fodder species were selected based on their traditional usage to treat diarrhoea and as dietary supplements for cattle. This research aimed to evaluate the nutritional composition and anti-nutritional factors as well as in vitro antibacterial, anti-biofilm, antioxidant, cytotoxicity, and anti-inflammatory activities of plant extracts against E. coli isolates from cattle and reference strains using molecular and phenotypic methods were investigated. The leaves and pods of leguminous (Vachellia, Senegalia, Dichrostachys, Ceratonia, Leucaena) and non-leguminous (Grewia, Morus, Salix, Searsia, Ziziphus) fodder species were collected in the Onderstepoort area, Pretoria North, Gauteng. ICP-MS (Inductively Coupled Plasma Mass Spectrometry), ICP-OES (ICP-Optical Emission Spectrophotometry) and standard methods were utilized to measure trace elements, proximate composition of fodder and tannins. Preliminary screening for antibacterial, antibiofilm (prevention 0 h and eradication 24 h), anti-aggregation and anti-motility activity against three ATCC strains (295225, 35218 and O157:H7) and cytotoxicity against Vero and Caco-2 cells was conducted to select the most active plant extracts for further analysis. Quorum sensing ability was explored using the biosensor strain Chromobacterium violaceum ATCC 12472. Thereafter, characterization of the isolates according to their E. coli UID A housekeeping gene, pathotypes EAEC (pCVD4), EPEC (eae), ETEC (st), EHEC (stx), DAEC (afa B-C), and detection of virulence genes such as agn43/flu, flic, luxS, flix A was undertaken using multiplex PCR. antimicrobial susceptibility testing (AST), anti-biofilm and anti-motility were investigated against ten E. coli strains isolated from healthy cattle faeces against different commercial antibiotics and active plant extracts from the preliminary screening. The seven plants that had good antibiofilm activities were selected for the determination of anti-inflammatory and antioxidant activities using colorimetric tests and qPCR. UPLC/MS/MS analysis was carried out to identify possible active phytochemicals responsible for anti-inflammatory and antibacterial activities of the methanol and acetone extracts of E. rigida. The results highlighted the presence of adequate levels of essential elements such as Zn (21.20-58.00 mg/kg), Co (0.06-3.75 mg/kg), Cr (0.5-5.08 mg/kg), Mn (9.02-197 mg/kg), Mg (0.10-1.08 mg/kg), Fe (42.40-812 mg/kg) and Na (72.00-1721 mg/kg). Notably, toxic elements including were found to be below toxic levels. Proximate analysis revealed the presence of dry matter (> 90%), ash (3.77-26.98%), CP (8.22-22.19%), carbohydrates (54.00-86.79%), fats (0.62- 4.53 mg/mg DM) crude fibre (10.54-40.10%) and neutral detergent fibre (NDF=22.26-59.20%). The gross energy (GE) ranged from 100 to 161.33 MJ/kg DM for most of the selected legume species. Lastly, tannin was present at a safe level (< 50 mg/kg) for ruminant consumption. The antibacterial results revealed that all plants had weak antibacterial activity (MIC > 0.62 mg/ml) with relatively low cytotoxicity (LC50 > 0.02 mg/ml). Approximately 28% of plant extracts eradicated established biofilms (24 h) by more than 50%. Interestingly, both methanol and acetone extracts of E. rigida were the only extracts able to prevent biofilm formation and to eradicate pre-formed biofilm by >50%, hence it was selected for further analysis. The half-maximal concentration of extracts inhibiting violacein production (ranging from 0.01 to 0.002 mg/ml) in all the selected plants with V. erioloba acetone exhibited the best results. E. coli O157:H7 is a hydrophobic and autoaggregating and highly motile organism, but these properties were significantly (p< 0.05) altered after treatment with plant extracts at a sub-MIC concentration of 0.16 mg/ml. Consequently, most plant extracts were inactive in decreasing the EPS production by altering polysaccharides, but the acetone extract of V. nilotica and S. galpinii slowed EPS production by 24% and 28% respectively. Molecular analysis results confirmed that 100% of the bacterial isolates were E. coli strains which were multidrug-resistant against ampicillin (100%), amoxicillin (40%), and tetracycline (30%). The E. rigida methanol extract was non-cytotoxic to Caco2 cells and showed good antibacterial activity against six E. coli isolates with MIC ranging from 0.16 to 0.31 mg/ml compared to acetone (1.25 to 2.5 mg/ml). The E. coli isolates did not harbor any selected genes associated with five diarrhoeal pathotypes including more importantly, the STEC, however, expressed presence of biofilm formation genes (fliA, fliC, (Ag43/flu)) and quorum sensing (lux). All strains were non-aggregative using the SAT test and highly motile with zones of migration < 20 mm after 48 h. E. rigida extracts had motility inhibitory effects in dose dose-dependent manner against E. coli isolates after 48 h incubation. Acetone and methanolic extracts of E. rigida (at 1 mg/ml) inhibited biofilm formation of the E. coli isolates by more than 50%. Seven plants with good antibiofilm formation activity against E. coli ATCC strains were further investigated for their anti-inflammatory and antioxidant activity. The methanol extracts of E. rigida V. tortilis and V. sieberiana inhibited NO release with IC50 values of 90.11, 101.52, and 94.11 μg/ml respectively in comparison to quercetin with IC50 = 30.00 μg/ml. Moreover, these extracts had high LC50 ≥ 70 μg/ml and were therefore relatively non-toxic to LPS-induced RAW 264.7 macrophages. E. rigida, V. sieberiana and V. tortilis extracts decreased the expression of iNOS, COX and IL-6 in LPS-activated macrophages in the same manner as the positive control quercetin. There was a positive correlation between iNOS mRNA expression and NO inhibition by the plant extracts and positive control quercetin. The plant extracts showed very strong antioxidant activity by ABTS and DPPH assays, and strong activity against 15-LOX with low IC50 values of 0.80 and 0.64 mg/ml respectively. UPLC-MS/MS analysis using both negative and positive ionization modes revealed the presence of (1) quercetin-3-O-α-L-rhamnopyranosyl-(1-->6)-β-D-galactopyranoside (609 m/z), (2) eupatin (359 m/z), (3) vicenin (m/z 293) (4) 9-oxo-10,12-octadecadienoic acid (293 m/z) and (5) isorhamnetin-3-O-rutinoside (623 m/z) in acetone and methanol extracts. These phytochemicals are classified as flavonoids and fatty acids which commonly have antimicrobial, antioxidant and inflammatory activities. In conclusion, the present study demonstrated that the methanolic extract of E. rigida, containing flavonoid compounds, essential trace elements (zinc and copper) and crude proteins, may be further investigated as supplementary animal feed or as a phytogenic feed additive because of its useful pharmacological activities including anti-diarrhoeal, antimicrobial, and anti-biofilm activities against multidrug resistant E. coli pathogens. The plant extracts were not toxic to mammalian cells at the highest concentration tested, but animal studies are required to confirm their efficacy because in vitro activity does not translate to in vivo potential. Moreover, the E. rigida extract has the potential to be formulated into antimicrobial surface cleaners and disinfectants to eradicate biofilms on various surfaces such as those in meat processing facilities to avoid contamination of human products.