In recent years, the incidence of both phenotypic and genotypic aminoglycoside resistance has been surging around the world [22, 23]. The corresponding high resistance rate can severely complicate combination therapy for aminoglycoside agents with broad-spectrum β-lactams including cephalosporins and carbapenems against severe Gram-negative infections, particularly in case of nosocomial infections [24, 25]. Since aminoglycoside agents are the first choice of clinicians for infection treatments  and given that aminoglycosides are the most commonly prescribed antimicrobial agents for treating serious GNB in Iran, an attempt is made here to characterize the aminoglycoside resistance by means of phenotypic and genotypic methods among five important GNB isolated from pediatric and general hospitals in Iran. The results of antimicrobial susceptibility screening revealed that about half of the GNB were fully resistant to at least one of the tested aminoglycosides, including gentamicin, tobramycin, and amikacin. Previously, resistance to gentamicin, amikacin, and tobramycin, which are considered as newer aminoglycosides with a broader spectrum of antibacterial activities, was generally found to be less common than resistance to older aminoglycosides such as streptomycin, kanamycin, and neomycin .
In the current study, approximately, more than 90% of Acinetobacter spp. exhibited resistance to all the tested aminoglycosides. In a previously published study by Aliakbarzade et al., 103 clinical A. baumannii strains were collected from Imam Reza Hospital of Tabriz University of Medical Sciences. They showed that A. baumannii strains were isolated from different clinical samples such as urine, sputum, tracheal secretion, bronchial lavage, wound, and blood. The findings of their study revealed that the rates of resistance to gentamicin, amikacin, and tobramycin were 86%, 81%, and 63%, respectively .
Compared to the above studies, we witnessed a significant increase in the number of aminoglycoside-resistant Acinetobacter isolates. In this study, the rate of resistance to gentamicin, amikacin, and tobramycin was 93.6%, 90.2%, and 90.5%, respectively. The high-level resistance to aminoglycoside is a serious issue for combination therapy of aminoglycoside with broad-spectrum β-lactams including carbapenems and cephalosporins against Acinetobacter infections . According to our findings, in comparison to other tested aminoglycoside agents, amikacin caused less resistance in GNB, especially in E. coli isolates, and is still an extremely useful drug in treating severe E. coli infections. Several studies have pointed out that increased resistance against amikacin could be associated with the unrestricted use of this compound by some clinicians [28, 29].
In 2018, Nasiri et al. surveyed the molecular epidemiology of aminoglycoside resistance in clinical isolates of K. pneumoniae. They collected 177 K. pneumoniae strains from the patients admitted to intensive care units (ICUs) as well as infectious diseases, internal medicine, and surgery wards. K. pneumoniae strains were isolated from different clinical specimens such as urine, wound, sputum, trachea, and blood. The above authors reported that amikacin was a more active antimicrobial agent than other aminoglycosides toward clinical isolates of K. pneumoniae with a resistance rate of 61% . Nevertheless, our findings show that the rate of resistance to amikacin in fact increased to 93.6% among K. pneumoniae isolates, compared to the mentioned studies. Overall, the high frequency of aminoglycoside-resistant GNB suggests that extensive use of these antimicrobial agents in clinical settings of Iran has led to the emergence of resistant isolates.
This study surveyed the prevalence of six main aminoglycoside-resistant genes in GNB. Results showed that a majority of aminoglycoside-resistant GNB (about three-quarters of isolates) harbored at least one AME gene. In total, the aac (6')-Ib (59%) and aph (3')-VIe (48.7%) genes were the most prevalent AME genes among all the aminoglycoside-resistant GNB. Related reports from different parts of the world have illustrated that aac (6´)-II and ant (2´´)-I genes are the most prevalent AME genes in Europe. Moreover, it has been revealed that aph (3′)-VIe, ant (2´´)-I, and aac (6´)-I genes have the highest frequency among AME genes in Korea [11, 30]. On the other hand, aac (6´)-31/aadA15 and aadA2 genes were also detected frequently in the GNB isolated from nosocomial infections in Mexico and Brazil [31, 32]. AMEs are the most important sources of aminoglycoside resistance among bacteria. The corresponding genes are highly mobile and can be transported by integrons, transposons, plasmids, and other transposable gene elements, often along with other resistant genes (such β-lactamases genes). As a matter of fact, the most threatening GNB acquire AME genes through horizontal gene transfer [33, 34].
In total, the aac (6') gene confers resistance to all of the aminoglycosides, except streptomycin. The aph (3′)-VIe was identified in A. baumannii and it conferred resistance to kanamycin, amikacin, neomycin, ribostamycin, paromomycin, butirosin, and isepamycin. The aph (3′)-II gene was described in the P. aeruginosa isolates. The aph (3')-II gene confers resistance to kanamycin, paromomycin, butirosin, neomycin, and ribostamycin. The aadA1 gene remains resistant to streptomycin and spectinomycin. Moreover, the aph (3')-Ia and aph (6) genes correspond to the resistance to kanamycin and tobramycin, respectively [17, 35, 36].
In Iran, a high prevalence rate of AMEs was previously reported in GNBs such as P. aeruginosa, A. baumannii, and K. pneumoniae [20, 37, 38]. The aph (3')-VIe was the most common AME in Acinetobacter isolates (52.6%), followed by aadA15 (37.9%), aph (3')-Ia (35.5%), and aac (6')-Ib (34.3%). In a study conducted by Aghazadeh et al. in Iran, aph(3')-VIe and aph(3')-II were the most prevalent AME genes in A. baumannii with prevalence rates of 90.6% and 61.8%, respectively . In another study in Iran, Asadollahi et al. reported that AME genes including aadA12, aacC1, and aadB were the most prevalent ones among A. baumannii . Altogether, these data indicate that aph (3')-VIe and aadA15 genes contribute to aminoglycoside resistance among clinical isolates of A. baumannii in different geographic locations of Iran. Our results were similar to those found by Nasiri et al. in Iran who reported aac(6′) as the most dominant AME among clinical isolates of K. pneumoniae . However, Ghotaslou et al. previously reported that ant(3″)-Ia and aph(3″)-Ib were the most prevalent AME genes in Enterobacteriaceae isolates in the northwest of Iran with frequency rates of 35.9% and 30.5%, respectively . In another research by Soleimani et al., aac (3)-IIa and ant(2″)-Ia genes were identified as the most common AMEs in uropathogenic E. coli isolated from an Iranian hospital . These data suggested that various reasons such as diversity of specimen type, geographic regions, sample size, bacterial sources, usage of antibiotics, and applied detecting methods would affect the distribution patterns of AME. Liang et al. previously reported that aac (3)-II, aac (6′)-Ib, and ant (3″)-I genes were the most common AME genes in K. pneumoniae isolates in China . In Norway, Lindemann et al. indicated that the majority of E. coli and K. pneumoniae isolates in their study harbored aac(3)-IIa and aac(6′)-Ib genes . The significant variation of the results may be attributed to geographical factors. Regarding P. aeruginosa isolates, we found aac (6')-Ib and aph (3')-II as the most prevalent AME genes. These findings are consistent with those reported by Aghazadeh et al. in Tabriz, Iran . In general, results demonstrated that more than 90% of GNB were resistant to one of the antibiotics. However, the results of the molecular method revealed that 59% of GNB harbored the aac (6')-Ib gene. Our analyses revealed that 78% of GNB were positive for at least one of the examined genes.
The limitation of this study is that we just sequenced one positive sample of each gene. For sequencing the aac (6')-Ib, aph (3')-II, aph (6), aadA15, and aph (3')-Ia genes, we used a positive sample in E. coli isolates. Moreover, one positive sample among A. baumannii isolates was used for aph (3')-VIe gene sequencing. This limitation was due to the budget limitation. In the analyzed sequences, all taxon affiliation was performed automatically by GenBank in the sequence submission process.