Genetic characterization and antimicrobial susceptibility of Escherichia coli isolated from household water sources in northern Ghana

Escherichia coli (E. coli) are bacteria that are commonly found in the lower gastrointestinal tract of humans and warm-blooded animals (Kittana et al. 2018). Most strains of E. coli are non-pathogenic, but some strains have acquired virulence factors and are capable of causing disease (Ramírez et al. 2013). E. coli can contaminate drinking water sources when indiscriminately excreted by humans and warm-blooded animals (Cabral 2010). Practices such as open defecation, the use of manure in agriculture, and inadequate drainage systems can result in the contamination of water sources with microbes such as E. coli after runoffs (Boelee et al. 2019).

The occurrence of E. coli in water is widely used as a bacteriological indicator of water quality and faecal pollution (Robert & Dirk 2017). The presence of pathogenic E. coli in water used for drinking, irrigation, and recreational purposes poses a potential health risk to humans and animals (Ramírez et al. 2013). Also, E. coli isolated from environmental waters can be multidrug resistant and of public health importance (Ramírez et al. 2013). Multidrug resistance here is defined as the acquisition of non-susceptibility to at least one antimicrobial agent in three or more antimicrobial categories (Magiorakos et al. 2012).

In the northern region of Ghana, of the 1.9 million inhabitants, about 50% of the people do not have access to improved drinking water sources and use unimproved water, i.e., surface water, as drinking water sources (United Nations (UN) 2020). This makes people vulnerable to waterborne diseases. Contaminated water and food have been attributed as the causes of diarrhoea (World Health Organization (WHO) 2017). Diarrhoea has been reported to account for the mortality of 525,000 infants annually (WHO 2017).

There are several etiologies responsible for infectious gastroenteritis causing acute diarrhoea (Shrivastava et al. 2017). However, diarrhoea outbreak among infants has largely been associated with pathogenic E. coli (Ochoa & Contreras 2011). Pathogenic E. coli capable of causing diarrhoea has been differentiated into enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAggEC), verotoxigenic E. coli (VTEC), diffusely adherent E. coli (DAEC), and enteroinvasive E. coli (EIEC) (Hien et al. 2007).

In Ghana, about half of all in-patients are exposed to one or more antibacterial therapy (Appiah-Korang et al. 2021), and about 90% of animal farmers purchase and administer antibiotics without prescription (Phares et al. 2020). Two separate multicentre point prevalence surveys involving four countries, including Ghana, and seven satellite hospitals in Ghana, respectively, revealed a high antibiotic use of 55 and 54.9% (Appiah-Korang et al. 2021; D'Arcy et al. 2021). In another survey, over 30% (125/400) had not received doctor's prescription during their last illness (Jimah et al. 2020). Antibiotics widely used in Ghana, in descending order of use, include metronidazole, amoxicillin/Clavulanic acid, ceftriaxone, cefuroxime, and ciprofloxacin (Appiah-Korang et al. 2021). About half of the antibiotics are mostly prescribed for the management of community-acquired infections, one-third for prophylaxis, and a tenth for no specifically documented indications (Appiah-Korang et al. 2021).

The inadequate availability of scientific data regarding the genetic characterization of E. coli from drinking water sources, the prevalence of diseases, infections and subsequent deaths caused by pathogenic E. coli (Ameer et al. 2021), and the rise in reported cases of antibiotic-resistance and pathogenic E. coli in Ghana is of great concern (García-Vello et al. 2020). It is, therefore, necessary to elucidate the prevalence, genetic variability, and antibiotic susceptibility pattern of E. coli isolates in surface water sources in the northern region of Ghana. This will provide important data for public health surveillance on the spread of pathogenic E. coli.

Fifty-two water samples, upstream and downstream, from 26 sources (Figure 1), from five districts of the northern region of Ghana [Gushiegu (n = 12), Karaga (n = 8), Saboba (n = 12), Savelugu (n = 8), and Zabzugu (n = 12)] were aseptically collected using sterile 100 mL thio-bags (Thermo Scientific, UK). All water samples were transported in an ice chest containing ice packs to maintain the cold chain and analyzed within 24 h.

Culturing of E. coli was carried out as previously described by APHA (2012) and Lupindu (2017); briefly, 100 μl (0.1 ml) of each water sample was pipetted onto MacConkey agar (HiMedia, India). The water was then uniformly spread on the agar using a glass spreader and incubated at 37 °C for 24 h. Colony-forming units for each water sample ranged between 30 and 300 after incubation.

Pink-red colony growths with bile precipitate on MacConkey agar were morphologically and presumptively identified as E. coli, and subsequently, the presumptive E. coli isolates were biochemically confirmed using indole and citrate tests. A red-pink colony with bile precipitate on MacConkey agar, which was indole-positive and citrate-negative, was identified as E. coli (Lupindu 2017). The confirmed colonies were then aseptically picked and streaked on nutrient agar and incubated at 37 °C for 24 h to obtain pure culture isolates. The pure isolates were then used for the genetic characterization and antimicrobial susceptibility test.

Multiplex polymerase chain reactions (PCRs) were carried out with eight primer pairs as previously employed by Hien et al. (2007) (see Table 1 for primer sequences and genes for strain determination).

Each pure E. coli isolate harvested from nutrient agar plates was suspended in 30 μl Tris buffer (pH 7.9) to 1 McFarland standard (3 × 10⁸ bacteria suspension/ml) and boiled for 30 min at 99 °C to obtain genomic DNA for PCR amplification. Two microliters of lysate from each isolate was used as a DNA template source in the PCR amplification. The PCR was carried out in a final reaction volume of 25 μl containing 2 μl DNA template, 12.5 μl Taq polymerase (OneTaq 2 × master mix buffer) (0.2 mM dNTPs, 1.8 mM MgCl₂, 20 mM Tris–HCI (pH 8.9), 22 mM NH₄Cl, 22 mM KCl, 0.05% Tween 20, and 0.06% IGEPAL CA-630), and 3.0 μM of forward and reverse mix (New England Biolabs). The PCRs were carried out using the peqSTAR 96 Universal Gradient thermal cycler (VWR, USA). The PCR cycling conditions were set at 5 min for initial denaturation at 95 °C, 45 cycles of 94 °C for 40 s (denaturation), 53 °C for 60 s (annealing), and 72 °C for 60 s (extension). The amplified PCR products were then analyzed on 2.0% (w/v) agarose gel and visualized under a UV transilluminator (Thermo Fisher Scientific, UK) after ethidium bromide staining.

The disc diffusion method, also called the Kirby–Bauer method as recommended by CLSI (2015), was employed for the antimicrobial susceptibility test. The following antimicrobial agents were used to test for antimicrobial susceptibility, such as ceftazidime, cefuroxime, gentamicin, cefixime, ofloxacin, augmentin, nitrofurantoin, and ciprofloxacin, based on the European Committee on Antimicrobial Susceptibility Testing breakpoints (EUCAST 2018). The diameter of the zone of inhibition of each antibiotic was measured from the back of the plate against a dark background using a ruler, graduated in millimeters.

All 52 water samples were found positive for E. coli.

Thirty-nine out of the fifty-two confirmed E. coli isolates from the water samples were found to carry virulence genes in different proportions (Figure 2).

Based on the pattern of detected virulence genes (Figure 2), E. coli isolates were characterized into four apparent strains (Figure 3). However, 46.15% of E. coli isolates that carried virulence genes were not characterized due to unusual banding patterns (Figure 3).

All the 52 E. coli isolates from each of the water samples were resistant to at least two of the essential antimicrobials used. However, multidrug resistance was recorded in 25/52 (48.1%) of the isolates. The pattern of susceptibility is shown in Table 2), and the proportion of multidrug resistance of E. coli to categories and individual antimicrobials are shown in Table 3 and Figure 4), respectively.

Access to safe drinking water is a human right (UN 2010). Drinking water is required to be free from E. coli (WHO 2017). In this study, however, a wide-spread distribution of E. coli was observed in drinking water sources. The widespread E. coli distribution in the water was attributed to runoff from environments with poor sanitation practices such as open defecation (Osumanu et al. 2019). The consumption of water contaminated with E. coli leads to the outbreak of waterborne diseases and infections, which subsequently results in morbidity and mortality in children and adults (Ameyaw et al. 2017).

The occurrence of pathogenic E. coli (75%) as identified in this study was higher than what was formerly identified in some African countries such as in Côte d'Ivoire (68%) (Kambire et al. 2017) and South Africa (67.5%) (Obi et al. 2004). The occurrence of pathogenic E. coli in the water sources is a result of poor drainage systems and the inability of the people to access improved sanitation (WHO & UNICEF 2000).

The frequency of virulent genes in this study varied greatly than what was found in South Africa (Ndlovu et al. 2015) and Côte d'Ivoire (Kambire et al. 2017). A study conducted in Southern Africa recorded aggR genes as the most predominant genes (69%) in Paarl and the Berge rivers, followed by iPaH (31%), stx or vtx (15%), and eae (8%). In Côte d′Ivoire, est genes were higher than elt genes (Kambire et al. 2017). However, the prevalence and predominance of elt genes as found in this study agrees less with the previous studies conducted in South Africa and Côte d'Ivoire, but agrees more with the studies conducted in the USA and in Egypt (Shaheen et al. 2004; Cho et al. 2018).

Four strains of potential pathogenic E. coli were identified and characterized, namely, VTEC, EPEC, ETEC, and EIEC. According to Lupindu (2017), VTEC, ETEC, EPEC, and EIEC are part of the intestinal disease-causing E. coli that cause various degrees of diarrhoea in warm-blooded animals such as humans. The main potential disease-causing E. coli strain as identified in the study, VTEC, is known to be the cause of diseases like the most deadly hemorrhagic uremic syndrome (HUS) and hemorrhagic colitis, which are mainly observed in both the elderly and children (Kuhnert et al. 2000). ETEC, the second most predominant strain in this study, is the causative strain for watery diarrhoea in humans (Lupindu 2017). EPEC and EIEC, on the other hand, are also the principal causes of diarrhoea in children under 2 years (Li et al. 2009) and dysentery-like diarrhoea with accompanied fever, respectively (Todar 2012).

The prevalence and predominance of potential disease-causing strains of E. coli as identified in this study conforms with the 2017 World Health Organization report, which opined that about 525,000 children die annually due to the consumption of contaminated food and water.

Of the 39 identified potential pathogenic E. coli isolates, 46.15% were not characterized into strains because of their unusual banding patterns. It remains unclear whether these are novel strains until further tests are carried out to fully elucidate the isolates.

The rise in multidrug resistance of E. coli is a global health concern. Here, the 52 isolates from the 52 water samples were resistant to more than one of the antibiotics used. The prevalence of antibiotic resistance to more than one antibiotic as registered in this investigation was far more than the 49.48% that was earlier reported by Odonkor & Addo (2018) in southern Ghana. However, the multidrug resistance in this investigation was novel according to the recent definition of multidrug resistance (Magiorakos et al. 2012). Although similar to the work of Odonkor & Addo (2018) where E. coli isolates were most susceptible to ciprofloxacin, nitrofurantoin, and gentamicin, a decrease in the levels of susceptibility of ciprofloxacin, nitrofurantoin, and gentamicin was, however, observed in the present study. A decrease in the antimicrobial susceptibility of ciprofloxacin and gentamicin was also reported by Adzitey et al. (2015). The multidrug resistance revealed in this study confirmed speculation by WHO (2018) that there may be a worldwide increase in the prevalence of multidrug resistance of E. coli strains. It is also worrying to note that, although nitrofurantoin and ciprofloxacin were the most effective antimicrobials against the E. coli isolates, some of the E. coli isolates posed resistance to these antimicrobials.

The multidrug resistance recorded in this study could be as a result of mutations and the dynamic ability of E. coli to exchange genetic-resistance genes through horizontal gene transfer (Fodor et al. 2020). This could have also been fueled by the overuse or misuse of antimicrobials in animals and humans without expert advice (WHO 2018). An inadequate knowledge of antibiotics and the inappropriate prescription of antibiotics to patients could also be contributory factors for the surge in multidrug resistance in E. coli in this study (Afari-Asiedu et al. 2020).

The susceptibility of the E. coli isolates to nitrofurantoin and ciprofloxacin might be a result of the reduced prescription and usage of these antimicrobials (Appiah-Korang et al. 2021). However, the high antibiotic resistance recorded against ceftazidime, augmentin, and cefuroxime could be attributed to their regular prescription and usage, inexpensive nature, easy access and both use and misuse of these antibiotics by some Ghanaians.

The WHO asserted that novel antimicrobial resistance patterns are emerging and increasing all over the globe, thereby threatening the treatment of common infectious diseases, a scenario that will present an incidence/prevalence of prolonged illnesses, disability, and death (WHO 2018).

Here, our data show good sensitivity of ciprofloxacin and nitrofurantoin against the E. coli isolates, since most of the isolates were susceptible to these antimicrobials.

The susceptibility of the isolates to nitrofurantoin could be due to the uniqueness of the antibiotic, characterized by a hydantoin ring with a nitro-substituted furanyl side chain that is metabolized within the bacteria to produce reactive compounds that are bactericidal (Howard 2007; Sauberan & Bradley 2018). Also, the susceptibility of the isolates to ciprofloxacin could stem from the fact that ciprofloxacin is an antitoxin bacteria-killing antimicrobial of the fluoroquinolone medicine class that is able to restrain DNA replication by repressing DNA-gyrase and bacterial DNA topoisomerase (Hooper & Jacoby 2016).

The study revealed a 100% prevalence of E. coli in the surface water samples, of which 75% were classified as pathogenic. These were further characterized into four strains, namely VTEC (17%), EPEC (13%), ETEC (8%), and EIEC (2%). Notably, 18 E. coli harbouring virulence genes with unusual banding could not be easily characterized. A sequencing of the isolate could help in the categorization.

The study also revealed a 48.1% multidrug resistance in 25/52 of the water samples when E. coli isolates from the samples were tested against eight antimicrobial agents belonging to five antimicrobial categories and listed as part of the WHO's list of essential medicines. The most resisted antimicrobial agents in this study were ceftazidime, augmentin, cefuroxime, and cefixime, while the susceptible antimicrobial agents were ciprofloxacin, nitrofurantoin, oflaxacin, gentamicin, and cefixime.

The observed surge in multidrug resistance isolates in this study, coupled with the decrease in susceptibility against nitrofurantoin and ciprofloxacin as compared to earlier studies, is indicative of the fact that E. coli isolates are gradually gaining resistance to these all important antimicrobials. This is despite the study revealing that nitrofurantoin and ciprofloxacin were the most effective antimicrobials against the E. coli isolates. Further works on the antimicrobial resistance mechanism of the isolates are essential for the understanding of multidrug resistance and the management of water-related E. coli infections.

The current study was conducted in a resource-limited setting. As a result, only PCR, using E. coli virulence gene markers, was used in the genetic characterization and molecular confirmation of the E. coli isolates. Therefore, whole genome sequencing and/or 16S rRNA of all or some of the isolates is recommended for the confirmation of identities in future studies.

The authors would like to thank the University for Development Studies for allowing the use of its Microbiology/Biotechnology laboratory in carrying out the laboratory sessions of this study.

The authors have no conflict of interest to declare.

This study was mainly funded by the authors.

Data cannot be made publicly available; readers should contact the corresponding author for details.