Isolation of bacterial strains

Water samples of Dujla River were collected approximately 200m, while sewage samples from the discharge point of effluent of waste pipes. Air samples were collected  using the MAS-100 Eco (Merck) on Petri dishes filled with CCA ES medium (Chromo Coliform Agar Enhanced Selectivity, Merck), according to [13]. Samples were cultured by direct streaking method on Eosin Methylene Blue and MacConkey agars, and then incubated for 18–24 h at 37°C [10]. The tests were carried out on triple sugar iron agar media, urea, and indole, methyl-red, Voges–Proskauer, and citrate (IMViC) media. Bacterial incubation in the triple sugar iron agar and urea media was carried out at 37°C for 18–24 h. IMViC test was carried out at 37°C for 48 h. Test results referring to E. coli were then stained with Gram stain to determine bacterial cell morphology.

Antibiotic Susceptibility Testing (AST)

The AST of bacterial isolated was done using Kirby Bauer disk diffusion methods as per the standard protocol from clinical laboratory standard institute (CLSI) guidelines 2024 [14].

Determination of the minimum inhibitory concentrations (MICs) of heavy metals

The broth micro dilution method was used to determine the MICs of heavy metals as described elsewhere [15].  In brief, all 100 samples of E. coli were cultured in tryptic soy agar medium overnight at 37 C. The bacterial suspension was diluted to a 0.5 McFarland standard with sterilized saline water. Seven heavy metals were selected, including cobalt (Co, CoCl₂), zinc (Zn, ZnSO₄), manganese (Mn, MnCl₂), chromium (Cr, CrCl₃6H₂O), cadmium (Cd, CdCl₂), copper (Cu, CuCl₂2H₂O) and lead (PbCl₂). The compounds were purchased from Sigma Aldrich (USA). MICs were determined as the lowest concentration of heavy metal that completely inhibited the growth of a strain after 18–20 h of cultivation at 35 C.

PCR amplification of HMRGs and ARGs genes

According to the manufacturer’s instructions, the PrestoTM Mini gDNA bacteria kit (Geneaid) was used to extract bacterial DNA.  A volume of 25 μl was prepared for conducting PCR reactions containing 4 μl template, 2 μl forward primer (20 μM), 2 μl reverse primer (20 μM), 12 μl MyTaqTM Red Mix (2×), and added ddH2O to 25 μl. Primer used in the study were listed in Table (1).  Thermal Cycler T100TM (Bio-Rad) was used to carry out PCR amplification.  Pre-denaturation was carried out at 95°C for 1 min. Amplification of 30 cycles consisted of denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, and extension.

Results

Antibiotic susceptibility profile

The resistance phenotypes of E. coli isolated from the fresh water Dujla river (n = 50), and wastewater (n = 50) were determined by measuring MICs to the 13 antibiotics listed in Table (2). Ninety-six percent of isolates from either river were resistant to one or more of the antibiotics tested. Seventy-one percent of all isolates were resistant to three or more antibiotics. Resistance to amoxicillin, trimethoprim/ sulfamethoxazole and ciprofloxacin was most frequent (71, 67, and 66% respectively of all wastewater isolates.

While resistance to cefotaxamin, Ceftriaxone, Cephalexin acid, and Cefoxitin was less frequent but still relatively high (54-57% of all isolates). Ciprofloxacin resistance (MIC > 4 μg/ml) was observed in 20 isolates of fresh water and 66 from wastewater. Seven of these isolates were resistant to amikacin and ciprofloxacin (MICs > 32 μg/ml and > 4 μg/ml, respectively) and were classified as uropathogenic strains. By comparing resistance frequencies in Dujla and wastewater isolates, we observed significant differences in total resistance to antibiotic. However, resistance to trimethoprim / sulfamethoxazole in isolates from wastewater was about eight times higher than that in wastewater isolates. In general, data show a high prevalence of antibiotic resistance in E. coli isolates from both sources. This is consistent with our previous studies of E. coli.

MIC of heavy metals

Sewage water E. coli  isolates show high difference (p value <0.05) in MIC for both copper and lead (1600 mg/L, 320 mg/L Respectively) while other heavy metal shows no significant differences, either in sewage or in fresh water, Table (3). Generally, the MICs of heavy metals for swage water E. coli isolates ranged from ≤30 mg/L to 800 mg/L. The MIC ranges for the seven selected heavy metals were as follows: Pb, ≤8–450 mg/L; Cr, 40–150 mg/L; Zn, 30–800 mg/L; Co, 25–100 mg/L; Cu, 80- 400 mg/L; and Cd, 10–45 mg/L.

For freshwater E. coli isolates, the lowest MIC found was ≤8 mg/L for Pb and the highest MIC was 300 mg/L for Cu. Table (3).

Prevalence of heavy metal resistance genes

Six HMRGs, including  chrA, pcoA, arsB, pbrT, cadD and czrc, were detected in E. coli isolates Figure (1). In sewage, the chrA gene was most widespread 67%, followed by arsb (48), pcoA (45%) then by nearly the same percentage of both pbrT and cadD genes (30 and 28% respectively), whilst the frequency of czrc was lowest (6 %). In fresh water, the frequency of arsB was the highest 17% and the frequency of czrc was lowest 1%. In general, frequencies of HMRDs in sewage water are much higher than corresponding ones of fresh water, Figure (1).

Prevalence of ESBL genes

Four ESBL genes, including CTX, CTX-M, bla_SHV, OXA, were detected in E. coli isolates Figure (2). In sewages samples, E. coli  isolates, showing prevalence ESBL genes ranged from 96- 23, and as follows: OXA (96.1%) > CTX-M (75%) > bla_SHV (70%) > CTX (54%).  On the other hand, freshwater E. coli  isolates, showing lesser percentages and as follows:  CTX (54%) > bla_SHV (70%) > CTX-M (75%) > OXA (96.1%).

Correlation between HMRGs and ARGs

As shown in Table (4), cadD showed significantly positive correlation with CTX (r=0.96), bla_SHV (r=0.947). pbrT shows correlation with CTX-M (r=0.95) and with bla_SHV.  arsB gene is correlated with both CTX-M and bla_SHV (r=0.94, and  0.95 respectively). pcoA gene has strong correlation with CTX-M (r=0.991) (r=0.980), finally, chrA gene shows no relation with any Args tested (p values greater than 0.05).

Figures & Tables

Discussion

The unsupervised consumption of antibiotics, excessive intake, and improper independent use are the major contributors for the emergence of antibiotic resistance bacteria which are already freely lived in our environment.  Heavy metal pollutants could help persist with these bacteria and emerging new ones. In the study, we examined the presence of heavy metal resistance genes among bacterial strains harboring ESBL genes collected from sources, sewage and fresh water.  The most important finding of this work was co-occurrence of antibiotic and heavy metal resistance elements among bacterial strains and their co-transfer to the host strain.  The 100 tested isolates had an elevated level of β-lactam antibiotics resistance.

The rise in antibiotic resistance could stem from changes in efflux or metal sequestration processes, likely due to a coordinated response triggered by both metal and antibiotic stress. Various genetic responses at the transcriptional and translational levels can be interconnected to create a unified reaction to these dual stresses [21]. However, heavy metal resistance capability when coinciding with antibiotic resistance can lead to an increase in antibiotic resistance and enhance their environmental stability. Antimicrobial-resistant E. coli showed high resistance to beta-lactams. Correlation study showed a strong connection between HMRGs and ARGs, which attributed to co-resistance, Wastewater likely contributes to this resistance due to the overuse of these antibiotics, resulting in antibiotic residues and the development of bacterial resistance.   

ESBL harboring E. coli  was found to be correlated with HMRGs. ESBL positive bacteria, found in open aquatic environments affected by human activities, have been globally reported. The genes responsible for ESBL production are associated with genetic elements that promote the spread of resistance [22-25], it may also be responsible for HMRGs too.

The origins of metals like mercury, cadmium, copper, and zinc vary, stemming from sources such as solid waste disposal [26,27]. These metals, as they accumulate in the environment, can reach critical levels that trigger a co-selection mechanism alongside antibiotic resistance [28]. Interestingly, the prevalence of heavy metal resistance genes exhibited a positive correlation with cadmium levels, indicating a significant role of this metal in selecting such genes. Our research findings indicate that at the highest concentrations, 10% of the samples exhibited resistance to cadmium, while 20% showed resistance to lead.

E. coli  isolate shows resistant to both antibiotics and heavy metals, which is a major concern for public health. These bacteria can survive in conditions with heavy metal levels way above what's considered safe. What's more concerning is that these bacteria are the same types that often cause infections in humans. The ones that are resistant to multiple drugs can also withstand high levels of heavy metals. It's like they've co-evolved to survive under stress of antibiotic and heavy metals at the same time. This suggests that pollutants like heavy metals might be helping these bacteria become even more resistant to drugs. It is also noticed that the heavier metals there were, the better these bacteria were at resisting them.

Author Contributions

Assel R. Kadhim conducted the laboratory work.  

Melad K. Mohammed designed the experiments.

Ahmed D. Jabbar wrote the manuscript and data analysis

Acknowledgments

The author is thankful to the Deanship of Graduate Studies and Scientific Research at University of Bisha for supporting this work through the Fast-Track Research Support Program.

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

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