The development of antimicrobial resistance genes (ARGs) in Enterobacteriaceae members has become a global health problem because antibiotic resistance (AR) leads to limitations in the treatment options thereby increasing the rate of morbidity and mortality [38]. Although carbapenem resistance (CR) among E. coli strains has been globally reported [39, 40, 41], there is a dearth of information on Carbapenem-resistant Escherichia coli (CREC) isolated from different environmental niches in South Africa. In the Eastern Cape Province, this is the first study that evaluates the genetic diversity among CREC strains using Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) genotyping. The sources of contamination and AR patterns may generally be understood by using specific molecular marker to analyze the clonal similarities among bacterial isolates recovered from different environmental niches.
Among the total amount of presumptive E. coli isolates (238), the quantity of positively confirmed E. coli isolates (192/83%) was unsurprisingly very high and this was similar to other studies by Blaak et al. [42] and Maamar et al. [43] with both reporting high incidence of E. coli isolates from farm environments. In another study by Araújo et al. [44], E. coli was isolated from irrigation water as well as vegetable samples and this is in line with our findings. This study provides an evidence of the occurrence of pathogenic microorganisms in the environment linking to other niches and our results show the occurrence of CREC in farm samples (Fig. 1).
The natural environment plays a major part in the emergence of AR pathogens as a result of the discharge of WWTP final effluents into receiving surface waters which may be used for domestic and irrigation purposes [45, 46]. The reuse of wastewater in agricultural settings also contributes microbial flora to the natural environment [47], for instance during farm practices, there is high tendency of microbes being transferred to farm animals hence, some pathogens (such as bacteria, parasites, viruses) often emerge as zoonotic in origin. Previously established pathogens associated with raw farm produce (including fruits and vegetables) may re-emerge as more virulent pathogens (particularly E. coli) after the acquisition of new virulence factors, including AR determinants [48]. In this study, our findings show that at least 4 categories of diarrheagenic E. coli (DEC) are recognized, namely ETEC, EPEC, EAEC and STEC having a total of about 21% from 40% pathogenic E. coli strains that were recovered. Our results are in line with the results of Canizalez-Roman et al. [49] that reported the prevalence of some DEC strains isolated from food samples.
In this current study, meropenem had the highest percentage resistance (70%), next to imipenem (64%). Interestingly, our findings are in line with a report by Kagambega et al. [50] conducted in Burkina Faso and this is an evidence of the detection of multidrug resistant (MDR) E. coli strains in sub-Sahara Africa. Our results show that at least one out of the 6 detected E. coli pathotypes exhibited phenotypic resistance against one of the 4 test carbapenems (doripenem, imipenem, meropenem, ertapenem) while ertapenem had the highest antimicrobial activity as also reported in a study by Kuzucu et al. [51]. Although, little is known about the spread and clinical relevance of carbapenemase-producing genes (CRGs) in Africa, studies of Manenzhe et al. [52] and Brink et al. [53] provided some reports that investigated various ARGs. In another study by Cakar et al. [54], it was also reported that CRGs were abundant in bacterial isolates of Enterobacteriaceae family. Moquet et al. [55] and Baroud et al. [56] both investigated the presence of blaOXA−48−like harbored by E. coli strains and these findings are in accordance with our results. However, blaOXA−48−like had the lowest percentage occurrence among the other selected CRGs that were screened for in our present study. Another study by Fischer et al. [57] revealed that E. coli harbored blaVIM−1 gene in their report, although this gene was not screened for in this present study.
In our study, farm samples (comprising soil, vegetables and irrigation water) had the lowest percentage occurrence (7%) of CRGs among the sample types which may be due to washing after harvesting, however our findings suggest a public health risk and probability of illness if raw vegetables are consumed without washing properly. Moreover, irrigation water had the lowest prevalence (4%) which may be due to the reduction in numbers as a result of environmental or climatic factors that can affect the survival of the microorganisms in the aquatic environments. Hospital effluents (32%) and WWTP final effluents (29%) are evidently proven hotspots of pathogenic microorganisms that may harbour ARGs. Furthermore, our study corroborates with one health strategies to address AR, through improving awareness and understanding of antimicrobial resistance (AMR) by effective communication, education as well as training. To determine the relative implications of AMR emergence and spread in food-animal production, there is a significant challenge due to the interconnectedness as well as interdependence of epidemiological pathways between humans, animals and the environment [58]. In our present study, hospital wastewater isolates cutting across the various E. coli pathotypes harboured all three carbapenem-resistance genes but blaNDM−1 occurred the most. Among the farm samples, only STEC strains harboured CRGs.
Some selected E. coli strains were investigated genetically with the use of ERIC-PCR. Osińska et al. [59] reported ERIC-PCR genomic fingerprinting technique as a vital tool for evaluating genetic relationships between bacterial strains isolated from environmental niches and some other studies reported ERIC-PCR genotypic diversity of multidrug resistant (MDR) bacterial strains isolated from different sources [60, 61], However, in this study we analyzed selected E. coli strains comprising some CREC with the use of ERIC- fingerprinting in order to evaluate the links that exist between strains isolated from different sources. ERIC-PCR has been demonstrated to be an effective method in determining the genetic diversity or relatedness among bacterial species by grouping clusters according to band sizes [62]. Some strains showed equal clusters but were isolated from different sources and this indicates clonal similarities between these strains which may be as a result of a possible link between final effluent and receiving water sheds. In light of the various inter- and intra-genotypic background of E. coli pathotypes, it is of concern to comprehend if there is a connection between the ERIC-genotype and the existence of pathogenic E. coli strains in poorly treated WWTP final effluent which is being discharged into receiving surface waters.
In our study, 7 clusters of ERIC-PCR were generated from 31 pathogenic E. coli strains (Figs. 3) at 95 per cent similarity cut-off value and exhibited genetic heterogeneity, but Jonas et al. [63] reported isolates with similarities above 70–80% which were eventually assigned to the same genotypes. Another study by Pusparini et al. [64] reported several unique clusters of ERIC in E. coli strains isolated from ice cube production sites which showed genetic diversity with 50 per cent cut-off value which is in contrary to the similarity cut-off value generated in this present study. On the basis of ERIC-PCR fingerprint, some of the isolates included in our study were genetically diverse. This is the most expected outcome as the pathogenic strains were isolated after being randomly collected from different environmental sources during sample collection which demonstrates that, the transmission might have occurred from clones of different origins. These findings corroborated with other studies [65, 66, 67]. Some other studies that used ERIC-PCR in E. coli isolated from other sources include Zhang et al. [68] who reported genetic diversity analysis of E. coli serotypes isolated from retail foods in China and Oltramar et al. [69] who reported genetic heterogeneity of E. coli isolated from pasteurized milk in Brazil. In our present study, river water samples were also collected and this is corroborated by a study by Lyautey et al. [70] who reported distribution and diversity of E. coli in river water samples collected in Canada.
Our results showed ETEC which harbored blaNDM−1 and blaKPC isolated from wastewater treatment plant (WWTP) final effluent having similar gene clusters as strains from river water samples. This may be as a result of discharge of final effluent into the river because both samples were collected from the same study area. A study by Shaikh et al. [71] reported genetic diversity in E. coli and another member of Enterobacteriaceae (Klebsiella pneumonia) which were both isolated from clinical samples; however, in our present study, clinical samples were not collected. Our present study showed varied genetic diversity between three strains of neonatal meningitis E. coli (NMEC) isolated from WWTP final effluent and river water samples and this is in line with a study by Levert et al. [72] who reported genetic diversity of extraintestinal pathogenic strains of E. coli. In this study, STEC had the highest number of ERIC-PCR genotypes and our findings are in contrary with the report by Moredo et al. [73] where ETEC reportedly had the highest EPIC-PCR genotypes. Studies by Yasir et al. [74] and Sun et al. [75] reported the genetic diversities in multidrug resistant ESBL-producing E. coli although, our study did not screen for ESBL in the E. coli strains nevertheless provides an opportunity for further analysis. EPEC strains were predominantly isolated from river water samples and our study corroborates with another report by Khare et al. [76]. Our current study used ERIC-PCR to identify the various sources and this is supported by Ibekwe et al. [77] who reported E. coli isolated from swine wastewater samples in the United States.
This is the first study reporting the genetic diversity of carbapenem-resistant E. coli strains isolated from different environmental sources in the Eastern Cape Province, with other studies by Montso et al. [78] and Chukwu et al. [79] reporting on the genotypic diversity of E. coli isolates recovered from farms and water samples respectively both in the North West Province.