Effect of stress, antibiotics and phytochemicals on verotoxic isolates of acinetobacter haemolyticus and escherichia coli obtained from water and wastewater samples

Abstract

Water related issues such as water treatment and distribution have become extremely important all over the world due to population growth, growing urbanization, health and environmental pollutions. Contamination of water bodies especially in Africa with antibiotic resistant bacteria strains is a cause for concern. Escherichia coli O157 H:7, and various strains of non O157 E. coli and Acinetobacter spp. are known for antibiotic resistance. Both bacteria are environmental organisms found coexisting together with high potentials of exchange of resistance genes. Despite the stress conditions confronting these bacteria in water, food and the human body, in the form of disinfectants, antibiotics, salts and the innate immunity, they appear to develop adaptive mechanisms that enable them survive and cause infection. This therefore necessitates the need for investigation of effective virulence factor-targeted control measures.

Culture of 62 water samples on Brilliance E. coli/coliform selective medium (BECSM, Oxoid), Eosin Methylene Blue (EMB) agar, or Baumann’s enrichment medium (BEM) and Leeds Acinetobacter Medium (LAM) for the isolation of E. coli and Acinetobacter spp. was carried out. Isolates were investigated for virulence factors, antibiotic resistance and transformation of resistance genes. The effect of oxidative stress exerted by 0.3% Crystal violet, 0.3% Bile salt, 4.0% NaCl, and 8% ethanol on some of the multi-drug resistant strains as well as the effect of stem back extracts of Curtisia dentata on verotoxin production by the verotoxic strains was also investigated.

Out of the 69 isolates of E. coli (including O26:H11, O55, O111:NM, 72 O126, O44, O124, O96:H9, O103:H2, O145:NM and O145:H2.) and 41 isolates of Acinetobacter spp. with 26 (53.06%) of the E. coli and 6 (14.63%) of the A. haemolyticus isolates producing verotoxins, and no A. lwoffii isolate produced the toxins. Twenty five - 25(35.23%), 14(20.30%) and 28(40.58%) of the E. coli isolates were positive for VTx1&2, Vtx1 and Vtx2 respectively, 49(71.015%), were positive for extended-spectrum beta-lactamases (ESBLs), 7(77.78%) for serum resistance, 57(82.61%) for cell surface hydrophobicity, 48(69.57%) for gelatinase production and 37(53.62%) for haemolysin production. While transformation occurred among the E. coli and Acinetobacter isolates (transformation frequency: 13.3 x 10-7- 53.4-7), there was poor curing of the plasmid genes, a confirmation of presence of stable antibiotic resistant genes (DNA concentration between 42.7-123.8 μg) and intra-genetic transfer of multidrug resistant genes among isolates.

Oxidative stress due to chemicals, salts, alcohol or freeze-thawing (blow temperature stress) exerted various degrees of lethality on E. coli isolates with some bacterial strains losing their potential to express virulence factors with time. There was however, generally insignificant (t test; P≤0.05) lethal effect against all the A. haemolyticus isolates, but crystal violet exerted the highest lethal effect on some individual isolates followed by ethanol, bile salt and NaCl. Isolates from wastewater demonstrated the highest rate of resistance compared to isolates from river water. The cell kill index (CKI) increased as temperature stress (-5; -18; and -28ºC) increased with time. But the rate of loss of expression of virulence factors or viability was slower in isolates from wastewater and abattoir compared to those from river water. Sixty percent of the E. coli isolates showed various levels of resistance to different antibiotics

(ampicillin (10 μg), cefuroxime, cephalexin, ceftazidime and tetracycline (30 95 μg in each case)) (multidrug resistance index (MDRI) values 4.20-5.60%). Relative inhibition zone diameters (RIZD) of C. dentata extracts against E. coli serotypes ranged between 8-28% (MIC, 100-2500 μg/ml), while against A. lwoffii and A. haemolyiticus, the RIZD values ranged between 10-28% (MIC, 100-850 μg/ml) and 6-28% (MIC 150-2500 μg/ml) respectively. However, higher MICs (MIC, 70-2500 mg/ml) were recorded for isolates with high MDRI values. Extracts demonstrated inhibitory action against the expression of both Vtx1 and Vtx2 genes in E. coli, A. haemolyticus and A. lwoffii. Saponins, tannins, glycosides, anthraquinones, flavonoids, steroids, phenols quinones, anthocyanins, amines and carboxylic acids were present in C. dentata. Ethanol root bark extracts consistently showed the highest DPPH radical scavenging activity (62.43%), total phenol content (TPH) (57.62 26 mg GAE/g) and reducing power (RP) (41.32%), followed by those of the stem bark and leaf extracts with the respective values of 54.68%, 37.77 mg GAE/g and 21.83%. The extracts also induced the leakage of Na+ and K+ 107 ions from both test bacteria.

Detection of virulence factors, antibiotic resistance genes and transformation among these isolates is a very significant outcome that will influence approaches to proactive preventive and control measures and future investigations. Resistant verotoxic A. haemolyticus could further complicate treatment in verotoxic food-borne or nosocomial infections. Induction of cationic leakage by extracts of C. dentata is an indication of one of its mechanism of action on bacterial cells. The plant can therefore be a good source of antibiotic substances for composition as antioxidants or antimicrobials with novel mechanism of action for the treatment of verotoxic bacterial infections.