Antibiogram and beta-lactamase genes among cefotaxime resistant E. coli from wastewater treatment plant

Abstract

Background: The World Health Organization (WHO) recently classified Enterobacteriaceae resistance to third-generation cephalosporin into the group of pathogens with critical criteria for future research.

Methods: A study to assess the antibiogram and beta-lactamase genes among the cefotaxime resistant E. coli (CREc) from a South African wastewater treatment plant (WWTP) was conducted using standard phenotypic and molecular biology characterization methods.

Results: Approximate total E. coli (TEc) concentration (log₁₀ CFU/mL) ranged between 5.7 and 6.8 among which cefotaxime resistant E. coli were between 1.8 and 4.8 (log₁₀ CFU/mL) for cefotaxime antibiotic concentration of 4 and 8 mg/L in the influent samples. Effluent samples, heavily influenced by the chlorination had only 0.3 log₁₀ CFU/mL of TEc. Fifty-one cefotaxime resistant isolates were selected out of an overall of 75 isolates, and subjected to a new round of testing, with a follow up of 36 and 48 isolates for both colistin and gentamicin, respectively as guided by initial results. Selected CREc exhibited resistance to amoxicillin-clavulanic acid (35.3%; n = 51), colistin sulphate (76.5%; n = 36), ciprofloxacin (47.1%; n = 51), gentamicin (87.5%; n = 48) and intermediate-resistance to meropenem (11.8%; n = 51). Extended spectrum-beta-lactamase genes detected, viz.: bla_CTX-M (52.6%; n = 38) and bla_TEM (84.2%; n = 38) and concurrent bla_CTX-M + bla_TEM (36.8%; n = 38), but no bla_SHV was detected. Carbapenem resistance genes, blaKPC-2 (15.8%; n = 38), blaOXA-1 (57.9%; n = 38), blaNDM-1 (15.8%; n = 38) were also detected. Approximately, 10.5 - 36.8% (n = 38) co-occurrence of two or more beta-lactamase genes was detected in some isolates. Out of the selected number (n = 30), 7(23.3%) were enterotoxigenic E. coli (ETEC), 14 (46.7%) were Enteroaggregative E. coli (EAEC), but no enteropathogenic E. coli (EPEC) was detected.

Conclusion: Resistance to cefotaxime and the presence of a wide range of beta-lactamase genes exposed the potential risks associated with these pathogens via occupational and domestic exposure during the reuse of treated wastewater.

Keywords: Beta-lactamase genes; Carbapenem; Cefotaxime; Ciprofloxacin; Wastewater; bla CTX; bla OXA-1.

Fig. 1

a Count [Log₁₀ (CFU/mL)] of Presumptive Total E. coli (TEc) and Cefotaxime Resistant E. coli (CREc) in summer Influent samples (b) Count [Log₁₀ (CFU/mL)] of other Coliforms and Cefotaxime Resistant Coliforms in summer influent samples. Legend: Inf SR-3 NA = influent sample at sampling occasion 3 without antibiotic, Inf SR-3 A4 = influent sample at sample occasion 3 with 4 mg/L of cefotaxime antibiotic, Inf SR-3 A8 = influent sample 3 with 8 mg/L of cefotaxime antibiotic; Inf SR-2 NA = influent sample at sampling occasion 2 without antibiotic; Inf SR-2 A4 = influent sample at sample occasion 2 with 4 mg/L of cefotaxime antibiotic; Inf SR-2 A8 = influent sample 2 with 8 mg/L of cefotaxime antibiotic; Inf SR-1 NA = influent sample at sampling occasion 1 without antibiotic; Inf SR-1 A4 = influent sample at sample occasion 1 with 4 mg/L of cefotaxime antibiotic; Inf SR-1 A8 = influent sample 1 with 8 mg/L of cefotaxime antibiotic. Figure 1: c Count [Log₁₀ (CFU/mL)] of Presumptive Total E. coli (TEc) and Cefotaxime Resistant E. coli (CREc) in Winter Influent samples (d) Count [Log₁₀ (CFU/mL)] of other Coliforms and Cefotaxime Resistant Coliforms in Winter Influent samples. e Count [Log₁₀ (CFU/mL)] of E. coli and other coliforms in effluent samples. Legend: Inf SR-4 NA = influent sample at sampling occasion 4 without antibiotic, Inf SR-4 A4 = influent sample at sample occasion 4 with 4 mg/L of cefotaxime antibiotic; Inf SR-4A8 = influent sample during sample occasion 4 with 8 mg/L of cefotaxime antibiotic; Inf SR-4 NA = influent sample at sampling occasion 4 without antibiotic; Inf SR-5 A4 = influent sample at sample occasion 5 with 4 mg/L of cefotaxime antibiotic; Inf SR-5 A8 = influent sample during sample occasion 5 with 8 mg/L of cefotaxime antibiotic; Eff SR-1 NA = effluent sample occasion 1 for E. coli without antibiotic supplementation; Eff SR-2 NA = effluent sample occasion 2 for E. coli without antibiotic supplementation; Eff SR-3 NA = effluent sample occasion 3 for E. coli without antibiotic supplementation; Eff SR-1 O-NA = effluent sample occasion 1 for other coliforms without antibiotic supplementation etc.

Fig. 2

MAR Index of the cefotaxime resistant E. coli and their percentage of isolates

Fig. 3

Pathotypes of selected cefotaxime resistant E. coli (n = 30)