Esherichia coli is a common species of bacteria connected with nosocomial infections involving, pneumonia, infections of urinary tract [1]. Aminoglycoside are important types of antibiotics utilized in the treatment of severe infections caused by bacteria of Gram-negative, including ESBL producing E.coli [2]. They prevent protein synthesis of bacteria by linking irreversibly to the bacterial 30S ribosomal subunit, thereby leading to death of the cell.

Resistance of bacteria to aminoglycoside may be because of chromosomal mutation as such as acquisition of mobile genetic factors (transposons, integrons and plasmid) having genes of resistance. Aminoglycoside resistance is mediated by three various ways, including reduce accumulation of an intracellular antibiotic, alteration of an enzymatic drug and the substitution of ribosomal proteins [3].

Production of AMEs is one of the most commonly happening way of resistance to the aminoglycoside among E.coli. Enzymes in the AAC groups inhibit aminoglycoside antibiotics by acetylation, APH can phosphorylate aminoglycoside and ANT cause aminoglycosides adenylation [4]. The genes encoding this enzyme usually are located on plasmids carrying genes for ESBLs.

Samples Collection

Across sections procedure on patients visited in the period of September 2024 to December 2024. 65 urine specimens were isolated from patients from period of research and the specimens were taken to the laboratory.

Isolation and Identification of E.coli

Microbiological standard diagnostic criteria were used to isolate and identify the clinical isolates of E.coli, which included colony morphology, Gram stain and conventional biochemical tests. The VITEK-2 automated system has been utilized to diagnose E.coli.

Aminoglycoside Susceptibility Testing

The isolates were screened for the sensitivity versus 3 various antibiotics disks of the class by utilizing technique of Kirby-Bauer disk diffusion which utilized to measure inhibition zones in agreement with the recommendations of laboratory and clinical Standards system [5]. 

Aminoglycoside Molecular Screening

This thesis was carried out to discover the dissemination of the aminoglycoside modifying enzymes among the XDR E.coli isolates taken from center of burn in Najaf during period of 3 months. The early detection of aminoglycoside resistance genes may inactivate the dissemination of these isolates of XDR E.coli on the future.

Aminglycosides-Resistant XDR E.coli Isolates

Present study showed that resistance rate of 23 isolates of XDR E.coli against aminoglycoside groups: Gentamicin, amikacin and tobramycin. However, the resistance profiles of the isolates show that 21 (91.3%) were resistant to 1 or more antibiotics of aminoglycoside. The summary of resistance profiles for all tested isolates is shown in Table 1 and Figure 1. Overall, isolates were divided into five phenotype groups. First group (A1), 13 (56.5%) isolates exhibited resistant to all aminoglycosides tested. Second group (A2), 6 (25%) isolates manifested resistant to gentamicin and amikacin but sensitive to tobramycin. Third group (A3), 1(4.3%) isolates were tobramycin and gentamycin resistance but susceptible to amikacin. Fourth group (A4), 1 (4.3%) isolate was resistant to gentamicin but susceptible to amikacin and tobramycin. In contrast, the fifth group (A5), 2 (8.3%) isolate was susceptible to all aminoglycosides tested.

In addition, PCR was used to screen all XDR isolates for the presence of the selected Aminoglycoside Acetyltransferases (AAC), aac(6')-IIb, aac(3)-II specific primers. The occurrence of aminoglycoside resistance genes among the isolates are shown in Table 2. However, present study found that one type of aminoglycoside resistant genes [aac(6')-IIb] were found in 20 (86.95%) isolates and 1(4.3%) of the XDR isolates investigated were positive for aac(3)-IIgene.

Aminglycosides Resistant among XDR E.coli Isolates

Although aminoglycosides represent a small fraction of antibiotic consumption in Iraqi hospitals as compared with β-lactam antibiotics and fluoroquinolones, they are still significant class of antibiotics in treating life threatening E.coli and other Gram negative bacterial infections. Therefore, detecting the level of aminoglycoside resistance is an important task. Some aminoglycoside remain active against many XDR E.coli isolates [6].

Table 1: Aminoglycosides Resistance Patterns of 23 XDR E.coli Isolates

Table 2: Distribution of Aminoglycosides-Resistance Genes in 23 XDR Isolates of E.coli

Figure 1: Gel of Agarose with red save stained of mono-plex PCR Amplified Product Isolates DNA of Isolates with aac(6')-Ib Primer. The Electrophoresis Used for 120 min at 65 volt. Lane (L) is Marker of DNA Molecular Size (10,000 –bp Ladder). (432 bp)

Present study also focused on resistance patterns to three antibiotics of aminoglycosides (gentamycin, tobramycin and amikacin) in isolates of XDR E.coli. According to the sensitivity testing, these result reported that 21 (91.3%) of XDR isolates of E.coli were found to be resistant to at least 1 of aminoglycosides tested (Table 1). The occurrence phenotypes of aminoglycoside resistance in XDR E.coli isolates were divided into five groups: (A1) resistant to all aminoglycosides tested (56.5%), (A2) resistant to amikacin and gentamicin (25%), (A3) resistant to gentamicin and tobramycin (4.3%), (A4) resistant to gentamicin (4.3%) and (A5) susceptible to all aminoglycosides tested (8.6%). An important striking feature found in present study is differences in aminoglycosides resistance phenotypes among isolates suggesting that aminoglycoside resistance in XDR isolates might be mediated by complex and multifactorial mechanisms. Despite these various resistance phenotypes, aminoglycosides are still valuable weapons in antimicrobial treatment, particularly in the management of life threatening infections of E.coli in Najaf hospitals.

In this study, occurrence of the selected Aminoglycoside Acetyltransferases (AAC), aac(6')-IIb, aac(3)-II were investigated. Present study show that 20 (86.95%) of XDR isolates harbored aac(6')-IIb gene and only 1(4.3%) harbord aac(3)-II gene (Table 2). This result is in accordance with data from Egypt [7] and Poland [8], where found aac(6')-Ib as one of the most frequently detected gene encoding ACC in clinical isolates of E. coli.

The study emphasizes the significance of aminoglycoside resistant E.coli isolates as a cause of UTI infection in Najaf City. The vast majority of XDR isolates exhibited resistant to at least 1aminoglycoside.This study showed elevated occurrence of aac(6')-IIb gene among XDR isolates.

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2. Shokravi, Z. et al. “Detecting the frequency of aminoglycoside modifying enzyme encoding genes among clinical isolates of methicillin-resistant Staphylococcus aureus.” BioImpacts, vol. 5, no. 2, 2015, pp. 87–91.

3. Ramirez, M.S. and M.E. Tolmasky. “Aminoglycoside modifying enzymes.” Drug Resistance Updates, vol. 13, 2010, pp. 151–171.

4. Lim, J. et al. “The genetic characteristics of multidrug-resistant Acinetobacter baumannii coproducing 16S rRNA Methylase armA and Carbapenemase OXA-23.” Journal of Bacteriology and Virology, vol. 43, no. 1, 2013, pp. 27–36.

5. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 30th Edn., CLSI Guideline M100-ED30, Clinical and Laboratory Standards Institute, Wayne, PA, 2020.

6. Pfaller, M.A. et al. “Ceftolozane/Tazobactam Activity against Drug-Resistant Enterobacteriaceae and Pseudomonas aeruginosa Causing healthcare-associated infections in the Asia-pacific region (minus China, Australia and New Zealand): Report from an Antimicrobial Surveillance Programme (2013–2015).” International Journal of Antimicrobial Agents, vol. 51, 2018, pp. 181–189.

7. El-Badawy, M.F., et al. “Molecular identification of aminoglycoside-modifying enzymes and plasmid-mediated quinolone resistance genes among Klebsiella pneumoniae clinical isolates recovered from Egyptian Patients.” International Journal of Microbiology, 2017, Article ID 8050432.

8. Ojdana, D., et al. “Genetic basis of enzymatic resistance of E. coli to aminoglycosides.” Advances in Medical Sciences, vol. 63, 2018, pp. 9–13.