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. 2024 Mar 15;25(4):126–133. doi: 10.1177/17571774241239780

Genotypic and phenotypic characterization of determinants that mediate antimicrobial resistance in Escherichia coli strains of clinical origin in South-Western Nigeria

Sharon Akinpelu 1, Abraham Ajayi 2, Stella Ifeanyi Smith 2,✉, Adeyemi Isaac Adeleye 1
PMCID: PMC11268240  PMID: 39055678

Abstract

Background

Multidrug resistant bacterial pathogens employ different mechanisms in evading the action of antibiotics. Multidrug resistance is wide spread among strains of Escherichia coli implicated in several infections including urinary tract infections, gastrointestinal infections, meningitis and bacteraemia.

Aim/Objective

This study investigates the antibiotic resistance profile, efflux pump activity and biofilm formation ability of E. coli strains isolated from clinical samples.

Methods

A total of 32 E. coli strains isolated from clinical samples were characterized and subjected to antibiotic susceptibility testing using standard methods. Isolates were screened phenotypically for biofilm formation and efflux pump activity. While molecular detection of genes encoding curli fimbriae and efflux pump activity was done by PCR.

Results

All 32 (100%) E. coli isolates were resistant to ceftazidime, cefuroxime, cefixime, amoxicillin-clavulanate, ofloxacin and ciprofloxacin. While 30 (93.8%) were resistant to gentamicin, 27 (84.4%) were resistant to cefepime and the least resistance of 15.6% was to imipenem. Efflux pump encoding gene tolC was detected in 13(40.6%) of the isolates, while 1(3.1%) harboured acrA gene. acrB gene was not detected in any of the isolates. Seven (21.9%) of the isolates were strong biofilm formers, while 5 (15.6%) and 20 (62.5%) were moderate and weak biofilm formers respectively. csgA gene was detected in all E. coli isolates.

Discussion

High antibiotic resistance of E. coli strains observed in this study is of public health significance. . It is therefore important to scale up efforts in regular monitoring of antibiotic resistance in both community and hospital settings.

Keywords: Antibiotic resistance, biofilm, efflux pump, Escherichia coli

Background

As the fight against antibiotic resistance continues to intensify, bacterial pathogens keep evolving strategies of evading antibiotic therapy. Resistance have been reported virtually across all classes of antibiotics including last-line intervention options (WHO, 2014). Several drivers including misuse of antibiotics in clinical settings, over use and self-medication have exacerbated this phenomenon most especially in Sub-Saharan Africa where surveillance and regulation is inadequate (Imanpour et al., 2017). Escherichia coli a member of the Enterobacteriaceae have been implicated in several infections such as meningitis, urinary tract infections, bacteraemia and gastrointestinal infections (Sarowska et al., 2019). Multiple studies have reported the ability of E. coli to resist antibiotics in several classes with a surge in resistance to fluoroquinolones, and third and fourth generation cephalosporins (Bidell et al., 2016; Singh et al., 2018). They do this by employing different mechanisms including biofilm formation and efflux pump activity.

E. coli form biofilm that consist of a bacterial community enclosed in a matrix of extracellular polymeric substances that shield them from extreme environmental conditions including, limiting their exposure to antibiotic treatment (Sharma et al., 2016). This ability to form biofilm is being modulated molecularly by several genes. The csgBAC operon in E. coli consists of such genes that code regulatory proteins which regulate curli fimbriae production that is key in biofilm formation (Sharma et al., 2016). E. coli has been implicated in infections associated with implanted medical devices such as prosthetic joints and grafts, intravascular and urethral catheters (Sharma et al., 2016). The implication of biofilm formation is not limited to mediating antibiotic resistance, it also plays role in virulence. Biofilm formation by bacteria has been demonstrated in vitro and in vivo by several studies. Eshima et al. (2023) demonstrated the in vivo formation of biofilm by E. coli and Candida albicans in silkworms.

Karigoudar et al. (2019) reported the detection of biofilm formation in antibiotic resistant uropathogenic E. coli strains isolated from urine samples of urinary tract infection (UTI) patients in India. Similarly, Abad et al. (2019) reported antibiotic resistance in E. coli strains that had high, moderate and low biofilm forming ability. The acrAB-TolC efflux pump system identified among members of the family Enterobacteriaceae including E. coli has found clinical relevance as they have been implicated in the mediation of multidrug resistance (Swick et al., 2011, Perez et al., 2007). Hence, this study employs phenotypic and molecular methods in evaluating the influence of efflux pump activity and biofilm formation in antibiotic resistance in E. coli of clinical origin.

Methods

Bacterial strains

A total of 32 clinical E. coli strains (12-urine, 4-blood, 4-stool, 5-sputum, 1-ear swab, 4-wound and 2-pus) were obtained from a hospital in Ogun State, Nigeria. Identity of isolates were re-confirmed using standard biochemical test including motility, indole, urea, citrate utilization and Kliger’s Iron Agar test according to Cheesebrough (2006)

Antimicrobial susceptibility testing

Susceptibility to antibiotics was determined by the Kirby-Bauer disc diffusion according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2019). Antibiotics used were imipenem (10 µg), ofloxacin (5 µg), amoxicillin-clavulanic acid (30 µg), cefexime (5 µg), nitrofurantoin (30 µg), ciprofloxacin (5 µg), ceftazidime (30 µg), cefepime (30 µg), gentamicin (10 µg) and cefuroxime (30 µg) (Oxoid, Basingstoke, UK). E. coli ATCC 25,922 was used as quality control. Overnight tryptone soy broth (Oxoid, Basingstoke, UK) culture of isolates were inoculated onto nutrient agar (Oxoid, Basingstoke, UK) and incubated at 37°C for 24 h. Cell suspension was made by emulsifying one or two colonies in phosphate buffered saline which was adjusted to 0.5 McFarland standard and applied to the surface of Muller-Hinton agar (Oxoid, Basingstoke, UK) using sterile swabs. After which antibiotic discs were then dispensed on the inoculated plates using a disc dispenser and incubated for 24 h at 37°C.

Phenotypic screening of isolates for efflux pump activity

The ethidium bromide (EtBr) cartwheel method of Martins et al. (2011) was adapted to determine efflux pump activity in E. coli isolates. Isolates (approximately 106 cells per mL) were streaked on Muller-Hinton agar (Oxoid, Basingstoke, UK) plates containing 0 mg/L, 0.5 mg/L, 1 mg/L, 1.5 mg/L and 2 mg/L concentrations of EtBr and incubated at 37°C for 24 h. After incubation, the plates were examined under a trans-illuminator fitted with a UV light (Cleaver Scientific Ltd, Rugby, UK). Fluorescence of isolates at different concentration of EtBr was recorded. Isolates in which fluorescence was detected at minimum concentration (0.5 mg/L) of EtBr were noted as not possessing active efflux pumps, while those in which fluorescence was not detected possessed active efflux pumps.

Assay for biofilm formation

Biofilm formation assay was performed by the tissue culture plate technique described by Stephanovic et al. (2000). A single colony of each isolate was inoculated into brain heart infusion (BHI) broth (Oxoid, Basingstoke, UK) with 2 % sucrose and incubated for 18 h at 37°C and 200 µL of bacterial suspension was loaded into the individual wells of 96-well microtiter plate. 200 µL of sterile BHI broth was used as negative control. After 24-h incubation at 37°C, the content of each well was discarded by inverting and gently tapping the plates. The wells were washed three times with sterile deionized water to remove non-adherent bacteria. The wells were air-dried for 45 min and 200 µL of 0.1 % (v/v) crystal violet solution was added to each well and incubated at room temperature for 45 min and wells were washed four times with sterile deionized water to remove excess stain. The incorporated dye was then solubilized using 33 % glacial acetic acid. The OD of stained adherent bacteria was measured at 650 nm using the Emax® Plus Microplate Reader (Molecular Devices San Jose, CA). The assay was performed in triplicate. Biofilm forming potential was categorized as previously described by Hassan et al. (2011) into strong (OD ≥0.108), moderate (OD 0.108–0.083) and weak (OD <0.083).

Molecular detection of efflux pump activity and biofilm formation genes

DNA was isolated according to Kpoda et al. (2018). acrA, acrB, tolC and csgA genes for efflux pump activity and biofilm formation respectively were detected by PCR. A 20 µL PCR reaction volume containing specific primers listed in Table 1 was used. The reaction mix consisted of 0.6 µL of each primer, 4 µL of 5X ready to use PCR Master Mix (Solis Biodyne, Estonia), 4 µL of DNA template and 10.8 µL of sterile water. PCR amplification parameter were: 30 cycles of initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing temperature of each primer at 40 s, extension at 72°C for 1 min and final extension at 72°C for 10 min. PCR products were separated by electrophoresis at 100 V for 60 min in 1.5 % agarose gel stained with ethidium bromide and visualized under ultraviolet trans-illuminator (Cleaver Scientific Ltd). A 100 bp DNA ladder (Solis Biodyne, Estonia) was used as a molecular weight marker.

Table 1.

Primer Sequences Used for the Detection of Biofilm Formation and Efflux Pump Encoding Genes.

Primer Pair Sequence (5’-3′) Size (bp) Annealing Temperature (°C) Reference
csgA-FWcsgA-RV 5′-GCAATCGTATTCTCCGGTAG-3′ 418 51°C Schiebel et al. (2017)
5'-GATGAGCGGTCGCGTTGTTA-3’
acrA-FWacrA-RV 5′-CTCTCAGGCAGCTTAGCCCTAA-3′ 1000 55°C Chakrabarty et al. (2016)
5′AACAGTCAAAACTGAACCTCTGCA-3′
acrB-FWacrB-RV 5′-GGTCGATTCCGTTCTCCGTTA-3′ 104 55°C Chakrabarty et al. (2016)
5′-ATGACGTTTACTTCCAGGTAG-3′
tolC-FWtolC-RV 5′-AAGCCGAAAAACGCAACCT-3′ 100 54°C Chakrabarty et al. (2016)
5′-GATGGTCACTTACCGACTCTG-3′
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Results

Antibiotic susceptibility testing

All 32 (100%) E. coli isolates were resistant to ceftazidime, cefuroxime, cefixime, ofloxacin, amoxicillin + clavulanic acid and ciprofloxacin. Thirty (93.8%) of the isolates were resistant to gentamicin, while 27 (84.4%) were resistant to cefepime. Low resistance was observed to nitrofurantoin 9 (28.1%) and imipenem 6 (18.75%) as shown in Figure 1.

Figure 1.

Figure 1.

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Percentage antibiotics resistance of E. coli clinical isolates KEY: CAZ: ceftazidime; CRX: cefuroxime; GEN: gentamicin; CXM: cefixime; OFL: ofloxacin; AUG: amoxicillin-clavulanate; NIT: nitrofurantoin; CPR: ciprofloxacin; IMI: imipenem; FEP: cefepime.

Detection of biofilm formation and efflux pump activity genes

Thirty one (96.9%) of E. coli strains phenotypically exhibited efflux pump activity as they did not fluorescence under UV light at 0.5 mg/L EtBr concentration as shown in Figure 2. However, acrA gene was detected in only 3% of the E. coli strains, while 13 (40.6%) of the isolates possessed tolC gene. However, none of the isolates harboured acrB gene. csgA gene was detected in all 32 (100%) E. coli strains as shown in Figure 3.

Figure 2.

Figure 2.

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Ethidium bromide cartwheel test showing E. coli strains positive for efflux pump fluorescence under UV illumination. Isolate 15 and 17 are negative.

Figure 3.

Figure 3.

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Gel image of amplification products of csgA gene showing positive bands. M: 100bp DNA ladder; Lane PC: positive control; Lane 1–32: E. coli clinical isolates with csgA gene; NC: negative control.

Biofilm formation potential

Seven (21.9%) of the E. coli strains were strong biofilm formers, 5 (15.6%) were moderate and 20 (62.5%) were weak biofilm formers. The E. coli strain (E3) that was a strong biofilm former displayed pan resistance to all 10 test antibiotics used even though it did not possess any of the assayed efflux pump genes. Majority of the isolates exhibited similar resistance pattern irrespective of their clinical sources as shown in Table 2. At least one isolate from various clinical samples were strong biofilm formers with the exception of the isolate from ear swab that was a weak biofilm former.

Table 2.

Efflux Pump Genes, Biofilm Formation Potential and Antimicrobial Susceptibility Profile of E. coli Isolates.

Isolate ID Source Biofilm Forming Potential Efflux Pump Genes/Biofilm Formation Gene Antibiotic Resistance Profile
Strong Moderate Weak acrA acrB tolC csgA CAZ CRX GEN CXM OFL AUG NIT CPR IMP FEP
E2 Blood ✓ - - - + R R R R R R R R S R
E3 Stool ✓ - - - + R R R R R R R R R R
E4 Pus ✓ - - - + R R R R R R S R S R
E5 Urine ✓ - - - + R R R R R R S R S S
E7 Wound ✓ - - - + R R R R R R S R S R
E8 Sputum ✓ - - + + R R R R R R S R S S
E9 Blood ✓ - - + + R R R R R R S R R R
E13 Urine ✓ - - - + R R R R R R R R S R
E14 Urine ✓ - - - + R R R R R R R R S R
E16 Urine ✓ - - + + R R R R R R S R R R
E20 Urine ✓ - - + + R R R R R R S R S R
E23 Stool ✓ - - + + R R R R R R S R S R
E26 Urine ✓ - - - + R R R R R R S R R R
E27 Pus ✓ - - - + R R S R R R R R S R
E29 Sputum ✓ - - - + R R R R R R S R S R
E30 Urine ✓ - - + + R R R R R R S R S R
E31 Urine ✓ - - - + R R R R R R S R S R
E32 Pus ✓ - - + + R R R R R R S R S S
E34 Sputum ✓ - - + + R R R R R R S R S R
E35 Wound ✓ + - - + R R S R R R R R S R
E36 Urine ✓ - - - + R R R R R R S R S S
E37 Urine ✓ - - - + R R R R R R R R S R
E38 Blood ✓ - - + + R R R R R R S R S S
E40 Wound ✓ - - + + R R R R R R S R S R
E43 Wound ✓ - - - + R R R R R R R R S R
E44 Urine ✓ - - - + R R R R R R R R S R
E45 Ear swab ✓ - - - + R R R R R R S R R R
E46 Sputum ✓ - - + + R R R R R R S R S R
E47 Sputum ✓ - - + + R R R R R R S R S R
E48 Stool ✓ - - - + R R R R R R S R S R
E49 Wound ✓ - - + + R R R R R R S R R R
E50 Urine ✓ - - - + R R R R R R S R S R
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Discussion

Pathogenic E. coli strains that have evolved and acquired the capability to resist antibiotics are now wide spread causing difficult to treat infections in both nosocomial and community settings (Donkor et al. 2019). In this study, E. coli strains isolated from clinical samples exhibited 100%, 100% and 84.4% resistance to fluoroquinolones and third generation cephalosporin, while displaying 84.4% resistance to fourth generation cephalosporin. Similar observation was made by Oladipo et al. (2015) who reported 82.9% resistance of E. coli clinical isolates to different generations of cephalosporins in South-Western Nigeria. Eghieye et al. (2018) also reported that 76.9% of E. coli strains isolated from two hospitals in Abuja Nigeria were resistant to ceftazidime. Resistance to imipenem a carbapenem antibiotic was observed in 6 (18.75%) of E. coli strains which was low compared to other β-lactam antibiotics. However, this level of resistance has a public health significance since imipenem is a last-line antibiotic (Pappa-Wallace et al., 2011). Other workers (Liang et al., 2014; De Oliveira et al., 2018; Pormohammad et al., 2019) have also reported the resistance of E. coli to imipenem though with low percentages. Of the 32 E. coli isolates, 9 (28.1%) were resistant to nitrofurantoin which is similar to the findings of Nwafia et al. (2019) who reported a slightly higher percentage (38.57%) resistance of E. coli strains isolated from a tertiary hospital in Enugu, Nigeria to nitrofurantoin. Bacteria pathogens possess several mechanisms with which they evade the action of antibiotics. Efflux pump activity is a key mechanism that limits the concentration of antibiotics that accumulates in a bacterium (Martins and Amaral, 2012). In this study 96.9% of the E. coli strains phenotypically exhibited efflux pump activity. However, majority of them did not possess the AcrAB-TolC efflux pump system. tolC gene was detected in 13(40.6%) of the isolates and only one of the isolate possessed acrA gene. It could be inferred that these findings more or less did not have overwhelming influence on the susceptibility pattern displayed by various isolates as E. coli strain E4 isolated from stool showed pan resistance to all 10 test antibiotics even when it did not possess any of the genes. Obviously the apparently low carriage of efflux pump is due to the fact that other efflux pump genes not screened may be responsible for the observed high resistance pattern. Igwe et al. (2019) in Zaria Nigeria reported the detection of seven efflux pump genes (mdfA, emrB, emrD, emrE, acrA, acrB and tolC) in antibiotic resistant E. coli strains isolated from UTI and diarrheic patients. Elsewhere, Kafilzadeh and Farsimadan (2016) reported a positive correlation of multidrug efflux pump acrA, acrB and tolC in relation to antibiotic resistance pattern in E. coli strains isolated from patients in Iran.

Beyond the possession of efflux pump systems by bacterial pathogens, biofilm formation has been found to mediate antibiotic resistance and protection against host immune system (Lebeaux et al., 2014). E. coli biofilm have been implicated in indwelling medical devices associated infection such as catheter-associated urinary tract infection (CAUTI) which are persistent and difficult to treat (Sharma et al., 2016). In this study, all E. coli isolates were biofilm formers with varying degree. As observed, a great percentage (62.5%) of E. coli strains were weak biofilm formers but they still displayed high resistance to antibiotics which was similar to both the resistance profile of moderate and strong formers. This assertion is in line with the observation of Cepas et al. (2019), who reported no relationship with pan-multidrug resistance and biofilm formation in pathogenic Gram-negative bacteria. However, the role of biofilm formation in exacerbating antimicrobial resistance cannot be overemphasized as it has been well documented by several workers (Chen et al., 2018; Lajhar et al., 2018; Risal et al., 2018).

As revealed in this study, having bacteria with efflux pump activity and the potential to form biofilm in any clinical setting poses a huge challenge to the treatment of infections (Shakibaie, 2018). It also facilitates the continuous transmission of pathogens as eradication in hospital environments could be difficult because biofilm formation limits the effectiveness of certain disinfectants employed in sanitization processes (Essiet et al., 2023).

This study showed high level of resistance to multiple antibiotics in clinical isolates of E. coli with the detection of few efflux pump genes. Although follow up studies is being undertaken to detect presence of more efflux pumps, high levels of resistance may not be completely dependent on efflux pumps, their ability to form biofilm could also be an underlying factor. It is therefore important to scale up efforts in regular monitoring of antibiotic resistance in both community and hospital settings in order to limit the dissemination of antibiotic resistant bacterial pathogen implicated in nosocomial and community acquired infections.

Limitations

The conclusion drawn from this study might be limited in scope since the sample size used was small and wild-type or mutant strains were not included in the study as negative control. Furthermore, E. coli strains were obtained from just a health centre in one location which may not provide a holistic representation of isolates from other locations.

Footnotes

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article..

Ethical statement

Ethical approval

Ethical approval for this study was obtained from the Institutional Review Board, Nigerian Institute of Medical Research with code number IRBB/19/033.

ORCID iDs

Abraham Ajayi https://orcid.org/0000-0001-6681-1795

Stella Ifeanyi Smith https://orcid.org/0000-0003-2163-1189

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