Characterization of plasmids carrying blaCTX-M genes among extra-intestinal Escherichia coli clinical isolates in Ethiopia

Abstract

CTX-Ms are encoded by bla_CTX-M genes and are widely distributed extended-spectrum β-lactamases (ESBLs). They are the most important antimicrobial resistance (AMR) mechanism to β-lactam antibiotics in the Enterobacteriaceae. However, the role of transmissible AMR plasmids in the dissemination of bla_CTX-M genes has scarcely been studied in Africa where the burden of AMR is high and rapidly spreading. In this study, AMR plasmid transmissibility, replicon types and addiction systems were analysed in CTX-M-producing Escherichia coli clinical isolates in Ethiopia with a goal to provide molecular insight into mechanisms underlying such high prevalence and rapid dissemination. Of 100 CTX-Ms-producing isolates obtained from urine (84), pus (10) and blood (6) from four geographically distinct healthcare settings, 75% carried transmissible plasmids encoding for CTX-Ms, with CTX-M-15 being predominant (n = 51). Single IncF plasmids with the combination of F-FIA-FIB (n = 17) carried the bulk of bla_CTX-M-15 genes. In addition, IncF plasmids were associated with multiple addiction systems, ISEcp1 and various resistance phenotypes for non-cephalosporin antibiotics. Moreover, IncF plasmid carriage is associated with the international pandemic E. coli ST131 lineage. Furthermore, several CTX-M encoding plasmids were associated with serum survival of the strains, but less so with biofilm formation. Hence, both horizontal gene transfer and clonal expansion may contribute to the rapid and widespread distribution of bla_CTX-M genes among E. coli populations in Ethiopian clinical settings. This information is relevant for local epidemiology and surveillance, but also for global understanding of the successful dissemination of AMR gene carrying plasmids.

Figure 1

Comparison of the percent antibiotic resistance displayed by 75 original isolates and the corresponding plasmid recipient strains. Recipient strains are based upon either the E. coli J53 AziR background and achieved via conjugal mating (transconjugates) or the E. coli HB101 background achieved via chemical transformation (transformants). The percent resistance of the different parent (dark grey bars) and recipient (light grey bars) isolates was according to the CLSI disk diffusion breakpoints. Resistance was defined as isolates with intermediate resistance and complete resistance based upon the size of the inhibition zone compared to the reference strains ESBL negative E. coli ATCC 25,922 and ESBL positive K. pneumoniae subsp. pneumoniae ATCC 700,603. Antibiotics tested were amoxicillin-clavulanate (AMC), cefotaxime (CTX), ceftazidime (CAZ),), cefepime (CEF), cefoxitine (FOX), ciprofloxacin (CIP), Amikacin (AMK), gentamicin (GEN), Meropenem (MEM), Sulphamethoxazole-Trimethoprim (SXT).

Figure 2

Frequency of plasmid replicon types originated from E. coli isolates obtained from clinical samples collected from healthcare centers in Ethiopia. Of 75 recipient strains receiving one or more plasmids via conjugation or transformation, 64 contained plasmids that could be typed by the chosen PCR-based method. IncF based replicon types were most identified.

Figure 3

Biofilm formation efficiency of pathogenic E. coli strains, isolated from Ethiopian patients. Data was generated from a minimum of three biological and three technical replicates for every isolate and plotted using GraphPad-5.0. One-way ANOVA with inbuilt Tukey’s multiple comparison test was applied to calculate statistically significance between control strain E. coli J53 (black bar) and Parental strains (‘P’, dark grey bars) and respective recipient strains (‘R’, light grey bar). P < 0.0001: ***P < 0.001: **P < 0.01: *P > 0.05: non-significant (ns).

Figure 4

Survival efficiency of pathogenic E. coli strains grown in the presence of serum. Survival properties of 10 parental clinical isolates (dark grey bars) and their corresponding recipient strains (light grey bars) and the control strain J53 (black bar) after exposure to active human serum for 0 and 3 h. The susceptibility to killing was calculated as follows: log kill = (log10 CFU per milliliter of initially added bacteria—0 h)—(log10 CFU per milliliter of bacteria surviving the incubation after 3 h). GraphPad-5.0 was used to plot data from a minimum of two biological replicates of every isolate. Means and standard errors of the results are shown. One-way ANOVA with inbuilt Tukey’s multiple comparison test was applied to calculate statistical significance between the corresponding parental and recipient strains. P < 0.0001: ***P < 0.001: **P < 0.01: *P > 0.05: non-significant (ns).