Due to limited therapeutic options, Acinetobacter baumannii accounts for a high mortality rate induced by opportunistic nosocomial infections. A. baumanni-induced infections include meningitis, wounds, ventilator- associated pneumonia, peritonitis, bacteremia, and soft-tissue infections (1, 2). Nosocomial infections resulting from Acinetobacter baumannii are an increasing concern around the world, as they are resistant to many antimicrobial agents and can survive in different hospital environments (2, 3). Due to MDR, at least three groups of antibiotics consisting of quinolones, beta-lactams, carbapenems, and aminoglycosides are ineffective on A. baumannii. Therefore, healthcare systems in Iran are facing a significant challenge (2, 4). Additional studies have reported various pan drug-resistant (PDR) A. baumannii strains (5, 6). Limited therapeutic options have made treating MDR A. baumannii strain infections difficult (2). To fight A. baumannii infections in hospitalized patients, aminoglycosides, whether taken alone or in combination with β-lactams, have been used and remain an essential tool in the fight against MDR strain infections(7). The specific interaction of antimicrobial agents with the 30S ribosomal subunit components, 16S rRNA, blocks translation initiation (8). Aminoglycoside- modifying enzymes (AMEs) such as aminoglycoside O-nucleotidyltransferases (ANTs) (5, 9), aminoglycoside N-acetyltransferases (AACs), and aminoglycoside O-phosphotransferases (APHs) in A. baumanni cause aminoglycoside resistance. APHs are correlated­ with increased gentamicin resistance (5). A. baumannii has been reported to have a variety of AMEs, consisting of phosphotransferase variants: APH (3')-I, APH (3')-II, and APH(2'')-Ib, as well as variants of the nucleotidyltransferases ANT(3)-I, ANT(4')-I, ANT(2'')-I and the acetyltransferases AAC(3)-I, AAC(6')-I, AAC(6')-II, and AAC(6')-III(10). In addition, methylation of 16S rRNA contributes to the inactivation of aminoglycosides (8). Hyper resistance to aminoglycosides, except streptomycin, is caused by ArmA, RmtA, RmtB, RmtC, and RmtD (8،9). Acinetobacter baumannii usually acquires aminoglycoside resistance genes via transposons, plasmids, or class 1 integrons (Int‑1) containing single or multiple gene cassettes (5, 6). Thus, these resistance genes can be transmitted to other bacterial species.
The genotyping of A. baumannii is an essential method for the specification of the connection between genetics and epidemiology. ERIC-PCR (Enterobacterial repetitive intergenic consensus polymerase chain reaction) is a reliable and fast method for determining the genetic diversity or clonality of A. baumannii isolates (11, 12).
We examined the aminoglycoside resistance genes (aph (2'')-Id, ant (4′)-Ia, ant (3'')-I, aac (6′)-Ib, aac (3)-I, aph (3′)-I, aph (2'')-Ib, aph (2'')-Ic). We used ERIC-PCR to molecularly type MDR A. baumannii gathered from ICU patients with an immune deficiency.
 

 
Table 2. Frequency of aminoglycoside resistance genes in A. baumannii isolates

Table 3. Patterns of aminoglycoside resistance genes among A. baumannii isolates

ERIC-PCR
ERIC-PCR produced multiple bands which were different in size ranging from 100to 3000 bp. The patterns are sorted into six types (A-F) and 20 subtypes. Band patterns illustrated that 16 out of 20 subtypes were distinctive ERIC-PCR clusters and four singleton isolates. 13% and 10% of sets were A3/A4 and B3, respectively, the most common among A. baumannii isolates (Table4, Figure 1).

Figure 1. Dendrogram of enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC–PCR) of A. baumannii isolates. Scale represents percentages of similarity.

Discussion

A. baumannii is known as the leading cause of nosocomial infections in hospitals as it can acquire a wide spectrum of antimicrobial resistance (2, 3). Limited treatment options have made MDR A. baumannii hard to cure in many environments (10, 16-18). Colistin and tigecycline are the ultimate treating choices (2). This work revealed that 100% isolates of A. baumannii were MDR that were in line with publications from Iran .Studies conducted in Iran (10, 19, 20). In concordance with these results, Safari et al., (2017) found 98% of A. baumannii isolates to be XDR (20). Resistance to Tobramycin and Gentamicin was seen in 67% and 63% of isolates, respectively. The high frequency of aminoglycoside-resistant A. baumannii in our study and previous reports may be due to acquiring AMEs (21). It has been reported that A. baumannii has a wide range of aminoglycoside resistance-related genes. Various risk factors are indicated to be related to the emergence of aminoglycoside-resistant A. baumannii, such as widespread use of antimicrobial agents and inappropriate prescription of aminoglycosides (10). Moreover, the aac(6′)-Ib, aph(2'')-Id, and ant(3'')-I genes showed high prevalence than other genes in this study. Furthermore, the co-existing aac(6′)-Ib and ant(3'')-I was discovered in 33% of isolates. Isolates of A. baumannii in China showed that aac(6′)-Ib and ant(3'')-I are high frequency genes associated with resistance to aminoglycosides found, supporting this study's results (22). According to Haldorsen et al., study (2014) aac(6′)-Ib enzyme could alter amikacin, even in isolates that were phenotypically susceptible to amikacin- of E. coli and Klebsiella spp. (23).
A study conducted by Helmy and Kashef (2017) illustrated aac(6′)-Ib gene in 84.4% of aminoglycoside-resistant Enterobacteriaceae (21). A gene cassette, aac(6′)-Ib, is mainly associated with insertion sequences like IS26 and truncated integrons, class 1 integrons, which probably explains its frequency among A. baumannii isolates (24). Our findings are in line with previous pieces of literature which reported the aac(6′)-Ib and ant (3'')-I as high frequency genes in isolates of A. baumannii (9, 10, 22). In contrast, aac(3)-I and aph(3′)-I genes were less common in this study (8, 22). Similar to this study's results, Xiao et al., (2014) also reported the lower frequency of aac(3)-I and aph(3′)-I genes (16).
The molecular methods for typing A. boumannii are the most critical procedures to determine the association between genetics and epidemiology. ERIC-PCR is a reliable and rapid technique for evaluating genetic differences or clonal relatedness of A. baumannii isolates (11, 12). The ERIC-PCR displayed 2-7 bands (100-3000 bp) based on our findings. The patterns produced by ERIC-PCR were sorted into six types (A-F) and 20 subtypes. They have implied 16 distinctive clusters and four singleton isolates. The most frequent clusters among A. baumannii isolates were A3/A4 (13%) and B3 (10%). In a study conducted by Viana et al., (2011), typing by ERIC–PCR showed a high range of clonal diversity (38 different clones), indicating that A. baumannii spreading depends on multiple factors not entirely attributed to a predominant clone (11). In agreement with our findings, Codjoe et al., (2019) have shown that ERIC-PCR typing of Gram-negative bacilli resistant to carbapenem generates some bands (1 to 8) with different sizes (50-800 bp). Among the 111 carbapenem-resistant (CR) isolates studied, 93 complex dissimilarities were detected, while 18 similar band patterns were observed in pairs (12). Additional studies indicated that A. baumannii showed genetic diversity and heterogeneity. Falah et al., (2019), by identifying 14 different ERIC patterns (ERIC-types) including 11 regular types and three unique types, reported clonal diversity in 80 A. baumannii isolates (25). This study was contrary to Ece et al., (2015) who reported a clonal relationship between strains of A. baumannii collected from a tertiary care center in Turkey. Their results revealed that many A. baumannii clusters in the ICU were the main clusters (26).

 

Conclusion

Our work indicated the abundance of aminoglycoside-resistant A. baumannii in patients in ICU, as well as the high frequency of aminoglycoside- modifying enzymes: aac(6′)-Ib, ant(3'')-I and aph(2'')-Id. ERIC-PCR has also illustrated an increased level of diversity in A. baumannii isolates. Thus, it can be a convenient method for investigating A. baumanni to determine its isolates' genetic differences or clonal relatedness in clinical environments.

 

Acknowledgements

This work as PhD thesis in Microbiology was supported by Department of Microbiology, Zanjan Branch, Islamic Azad University, Zanjan, Iran.

 

Conflicts of Interest

The authors declare that they have no competing interests.