Introduction

Antibiotics have been the basis of therapy for bacterial infections since their emergence in the early 20th century. Nevertheless, the widespread development of resistance has created significant challenges. Multidrug resistance is characterized by the lack of susceptibility to at least one antimicrobial agent across three or more drug classes, as determined through in vitro susceptibility testing (1) . In Iraq, antibiotic abuse in hospitals and among farm animals is relatively frequent and common, as it is in many other nations. The uncontrolled and irrational use of drugs has increased multidrug-resistant (MDR) strains. Moreover, multidrug-resistant Escherichia coli (MDR) strains have been extensively studied in various regions (2-6). A recent study conducted in Basrah, Iraq, revealed that 58 (93.54%.) out of 62 samples of E. coli, isolated from environmental sources and cases of diarrhea, exhibited resistance to a minimum of three distinct antibiotics (7) . The results showed high resistance to penicillin, erythromycin, and tetracycline. The resistance rates were 62 (100%), 57 (91.93%), and 50 (80.64%) for each drug, respectively. Tests on sewage samples in Kuwait showed high resistance to cephalosporin antibiotics. They also found genes for Extended Spectrum Beta Lactamase (ESBL) and well-known strains like ST131 and ST648, which could pose health risks to the public (8) ., free-range hens in the Caatinga biome of Brazil have been found to harbor cephalosporin-resistant E. coli, particularly prevalent in Paraíba. These bacteria exhibit genetic diversity among various taxonomic classifications and possess genes that code for the CTX-M or AmpC enzymes (9) . Moreover, recently published research reported that 90% and 57.9% of E. coli strains isolated from diarrheagenic animal and human sources, respectively, exhibited a high prevalence of multi-drug resistance, particularly to ampicillin, cefoxitin, imipenem, and ciprofloxacin (10). Additionally, research conducted on clinical isolates from patients in Nigeria demonstrated a notable proportion of E. coli strains showing complete resistance to tetracycline and ampicillin, along with over 90% resistance to various other antibiotics evaluated, including levofloxacin, cefuroxime, ciprofloxacin, tobramycin, and amoxicillin/clavulanic acid (11). The spread of multidrug-resistant Escherichia coli found in clinical and environmental samples has to be continuously monitored despite these investigations. Therefore, the present study was designed to investigate and identify the frequency of multi-drug-resistant Escherichia coli isolating from diarrheal cases and environmental sources in Basrah, southern Iraq.

Materials and Methods

Sample collection:

From early December 2023 to July 2024, two hundred fifty samples were collected from different regions of Basrah province. The samples were distributed as follows:

Cattle sample: One hundred Fecal samples (1 to 2 grams each) were collected from the veterinary medicine hospital in Basrah province.

Human sample: One hundred stool samples (1 to 2 grams) were collected from the Al-Sadr Teaching and Republican Hospital in Basrah province.

Environmental sample:

Fifty environmental samples (1L) from water and (1gm) from soil farms in Basrah province from December 20230to July 2024.

All samples are collected under sterile conditions and immediately transparently in a cooling box to the Microbiology Laboratory at the College of Veterinary Medicine, Basrah University.

Culturing diagnosis step

The collected samples were first ed on MacConkey agar, subculturing on MacConkey agar, and subculture on Eosin methylene blue (EMB) agar. This step helps to isolate and differentiate the enteric bacteria. The agar plates inoculated with suspected bacteria were then placed in an incubator at a suitable temperature (37°C) for 24hr. Subsequently, the bacterial colonies with different morphologies (size, shape, color, texture) are observed on the agar plates culture to primarily select the suspected colonies for the successful analysis step.

Molecular diagnosis step

DNA Extraction Spin Kit from Anatolia company (Turkey) was used to extract the bacterial DNA based on a silica membranning column that facilitates the extraction and purification of bacterial nucleic acids (12). The traditional PCR was subsequently performed to detect the uidA gene, which encodes the β-glucuronidase enzyme, across all E. coli species. A specific foreword primer (F: 5-CCAAAAGCCAGACAGAGT-3) and reverse primer (R: 5-GCACAGCACTTCAAAGAG-3) were employed to achieve this goal. A total volume of 25 μl of PCR reaction tubes was prepared and consisted of 12.5 μl of premix hot-start enzyme, 1 μl (10 pmol) of each forward and reverse primer and 4 μl of DNA template. Subsequently, the final volume was accomplished by adding nuclease-free water. A PCR apparatus was used for the PCR amplification steps, starting with a 5-minute pre-PCR heating step at 95°C. Then 35 cycles, each consisting of one minute at 94°C, one minute at 58°C, and one minute at 72°C, then another 72°C for five minutes as a final extension (13). The size of the amplified product was verified utilizing a red-safe DNA solution in a 2% agarose gel electrophoresis (14).

Antimicrobial sensitivity test

Using the conventional Kirby-Bauer disc diffusion method outlined in the Clinical and Laboratory Standards Institute (CLSI) recommendations, all presumed pure colonies of E. coli were tested for their susceptibilities against the selected antibiotics (15). As indicated in Table 1, the bacterial isolates were examined for antibiotic resistance to six antibiotics from five distinct classes.

Results

Culturing diagnosis results

Both traditional and molecular approaches were used to detect E. coli in collected samples. The culturing results showed that 53(30 from cattle, 12 from humans, and 11 from the environment) exhibited a distinctive greenish metallic sheen appearance on EMB agar, while on MacConkey agar, the suspected colonies were growing to have a rounded, non-mucoid, bright pink (lactose fermenting) texture.

Molecular diagnosis results

The typical PCR technique was used to detect the presence of the uidA gene in the suspected E. coli isolates, and the results revealed that 53(100%) of isolates carried the target gene (Figure 1).

Figure 1.PCR products of the uidA gene of E. coli. The size of the PCR product is 623bp. M: Marker DNA ladder (100bp-3000bp).

Antimicrobial susceptibility testing results: A total of 53 E. coli isolates were examined for antibiotic resistance, comprising 30 from cattle, 12 from humans, and 11 from environmental sources. The assessment used the Kirby-Bauer disk diffusion method against six antibiotics from five distinct classes. Among the 30 isolates from cattle, varying levels of resistance were observed: 100% exhibited resistance to amoxicillin-clavulanic acid, which belongs to the penicillin class; 66.66% and 50% showed resistance to gentamicin and streptomycin, respectively, both of which are aminoglycosides; 56.66% were resistant to trimethoprim, associated with the dihydrofolate class; and 66.66% demonstrated resistance to tetracycline, categorized under tetracyclines (Table 2) and (Figure 2).

Figure 2. The percentage of MDR Escherichia coli isolated from cattle samples

On the other hand, Table (3) and Figure (3) demonstrate the results of MDR E. coli in 12 human isolates. The results showed varied levels of resistance to antibiotics, in which 100% of isolates was resistant to amoxicillin-clavulanic acid, which represents the penicillin's class. In comparison, 83.33% and 75% of isolates were resistant to gentamicin, streptomycin related to aminoglycosides class. In addition, 66.66% of isolates were resistant to tetracycline representing the tetracyclines class and 50% of isolates were shown resistance to trimethoprim related to dihydrofolate class. Moreover, the results of the antibiotic resistance susceptibility test of E. coli isolated from 11 environmental samples were showed also different levels of resistance in which 100% of isolated bacteria were resistant to amoxicillin-clavulanic acid and trimethoprim, which related to penicillin's and dihydrofolate classes. In contrast, 72.72% and 63.63% of isolates were resistant to gentamicin, streptomycin is related to aminoglycosides (Table 4) and (Figure 4).

Figure 3.The percentage of MDR Escherichia coli isolated from human samples

Figure 4. The percentage of MDR Escherichia coli isolated from environmental samples

Furthermore, Table (5) and Figures (5) and (6) revealed the incidence rate of Escherichia coli isolated among cattle, humans, and the environment, which seem to have similar rates of resistance for the five classes of antibiotics and antibiotic resistance profile. Table (6) presented the antimicrobial resistance pattern of E. coli that recovered from cattle, humans, and environment. These isolates were distributed into 31 patterns. he most commonly observed resistance patterns were NA - AMC - GN - S - TE - TR. The highest multi-resistance was observed, with 20.75% of the isolates (11 in total) exhibiting resistance to six different antimicrobials (Table 6).

Discussion

Antimicrobial resistance within the Enterobacteriaceae family, especially E. coli, poses a global health risk (16). Furthermore, the risk of E. coli is increasing, as this bacterium can be transmitted from animals to humans through direct contact. This is particularly evident among workers in large animal slaughterhouses, those with occupational exposure on farms, and through ingesting contaminated food. Moreover, E. coli can also be transmitted to humans indirectly through environmental routes (17).

Figure 5.The percentage of MDR Escherichia coli isolated from cattle, humans and the environment.

Figure 6.The correlation of MDR Escherichia coli isolated from cattle, humans and the environment with 5 antimicrobial agents.

In the current investigation, the findings from bacterial isolation indicated that among a total of 100 fecal samples collected from cattle, 100 stool samples from humans, and 50 environmental samples, 53 isolates demonstrated unique greenish metallic sheen colonies on EMB agar, along with rounded, non-mucoid, bright pink colonies exhibiting lactose fermentation on MacConkey agar. Additionally, the suspected bacterial isolates were identified through a conventional PCR assay targeting the uidA gene, which is universally present in all E. coli species. All isolates (100%) exhibited positive results, which agrees with the results of (18) . However, our research indicates a higher percentage of E. coli isolated from animals compared to previous studies, which reported isolation rates of 4.7% and 5% (19) . These findings could be attributed to the difference in findings between studies due to the use of dairy farm management measures, which prevent harmful bacteria from spreading throughout dairy cows. Cleaning the farm floor, all tools used, and providing safe water for dairy farms are important to prevent E. coli from growing and spreading among cows, which helps reduce the risk of bacterial contamination and infection (20) . On the other hand, the results demonstrated that the local Escherichia coli isolates recovered from cattle, humans, and the environment had similar rates of resistance to antibiotics for the five classes of antibiotics. This finding was consistent with (10), who discovered that human isolates demonstrated high resistance MDR patterns and that resistance to specific antibiotics might be attributable to exclusively treating humans and animals with that particular antibiotic. Moreover, (7) their study shows a multi-significant frequency of antibiotic resistance in E. coli that is isolated from diarrheal cases in both humans and animals. Antibiotic resistance presents a global challenge that poses significant risks tohumans' and animals' health and well-being (21).

A report by (22) indicates that if this issue remains unaddressed, it is projected that by 2050, antibiotic-resistant bacteria could result in an annual increase in mortality rates, potentially affecting up to 10 million individuals. Pathogenic bacteria exhibiting resistance can emerge in humans, animals, and the environment, posing a threat to public health due to the imprudent use of antibiotics, including both misuse and overuse, across different sectors such as livestock and human environments. In addition to antibiotic resistance in pathogenic bacteria, it has also been identified in commensal bacteria, including E. coli. This point of view is also shared by (23), which indicates that one of the commensal bacteria, E. coli, has developed resistance to more than one type of antibiotic. (24) found that E. coli can transfer antibiotic-resistant genes to other bacteria, including dangerous bacteria (25). On the other hand, bacteria that are resistant to at least one antimicrobial agent in three or more antimicrobial groups are known as multi-drug resistant (MDR) microorganisms. The current research demonstrated a rise in multiple drug resistance (MDR) across more than one antibiotic within five distinct antimicrobial categories. The findings indicated that the Escherichia coli isolates obtained from cattle, humans, and environmental sources exhibited comparable rates of antibiotic resistance across the five classes. Our research aligns with the findings of the study (26), which reported that almost 90% of isolates were multidrug resistant against at least three antibiotics and were identified in diarrheal samples collected from cattle farms in Pakistan. This study also indicated a significant level of antibiotic resistance, predominantly to (100%), ampicillin (100%), sulfamethoxazole-trimethoprim (85%), and tetracycline. Furthermore, the prevalence of multidrug-resistant E. coli was 57.3%, characterized by 39 distinct resistance patterns.

Conclusions

In conclusion, the emergence of pathotypes and multidrug-resistant E. coli infection in livestock is considered a public health issue in Basrah province.

Conflicts of interest

The authors declare that there is no conflict of interest.

Ethical Clearance

This work is approved by The Research Ethical Committee.

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