Frequency of Aminoglycoside-Modifying Enzymes and ArmA Among Different Sequence Groups of Acinetobacter baumannii in Iran

Abstract

We evaluated aminoglycoside resistance in 87 Acinetobacter baumannii strains isolated from four hospitals located in the North West region of Iran and typed them in sequence groups (SGs) using trilocus sequence-based scheme to compare their clonal relationships with international clones. Resistance toward aminoglycosides was assayed by minimum inhibitory concentration (MIC) and presence of aminoglycoside-modifying enzymes (AMEs), and ArmA-encoding genes were evaluated in different SGs. The majority of isolates belonged to SG1 (39%), SG2 (33.3%), and SG3 (12.6%), whereas the remaining ones were assigned to six novel variants of SGs. MIC determination revealed netilmicin as the most and kanamycin as the least active aminoglycosides against all groups. Among the varied SGs, isolates of SG2 showed more susceptibility toward all tested aminoglycosides. APH(3′′)-VIa-encoding gene was predominant in SG1 (47%), SG2 (62%), and SG6–9 (100%). However, AAC(3′)-Ia (100%) and ANT(2′)-Ia (90.9%) were the dominant AMEs in SG3. There was significant association between harboring of aminoglycoside resistance genes and specific aminoglycosides: gene encoded by APH(3′)-VIa was allied to resistance against amikacin and kanamycin, whereas ANT(2′)-Ia was related to the resistance toward gentamicin and tobramycin in SG2. In SG1, tobramycin resistance was correlated with harboring of AAC(6′)-Ib. Screening of armA demonstrated the presence of this gene in SG1 (58.8%), SG2 (10.3%), as well as SG3 (9%). Our results revealed definite correlation between the phenotypes and genotypes of aminoglycoside resistance in different clonal lineages of A. baumannii.

Introduction

Acinetobacter baumannii has indeed emerged as an etiologic concern for diverse nosocomial infections, including ventilator-associated pneumonia, catheter-related bloodstream, and urinary tract infections.³

The microorganism's ability to survive in a hospital setting and spread among patients along with their resistance toward multiple antibiotics are the main driving forces behind the frequent large outbreaks in different countries.¹⁴ Till date, globally, three epidemic international clones, namely I, II, and III have been reported to be responsible for most of the outbreaks.^8,37 However, other clone complexes corresponding to international clones have been found geographically disseminated with significant contribution.^7,34

The propensity for developing resistance to multiple classes of antimicrobial agents is a striking feature of strains belonging to these international clones. Although increasing resistance to carbapenems is being emphasized and well known in these strains, nonsusceptibility to traditional antibiotics like aminoglycosides is also a concern,²³ since these antibiotics have long been used for the treatment of life-threatening infections, especially in hospitalized patients. However, their efficacy has been reduced by the surge and dissemination of resistance.³⁹ The main factors contributing to the increasing resistance to aminoglycosides has been the genes encoding aminoglycoside-modifying enzymes (AMEs) classified as acetyltransferases (AACs), nucleotidyltranferases (ANTs), and phosphotransferases (APHs), usually found on plasmids or transposons, which facilitate acquisition of resistance phenotype.³⁹ The prevailing nomenclature system for these enzymes consists of a three-letter abbreviation identifying the enzyme activity, followed by the site of modification or regiospecificity in parentheses, a roman number particular to the resistant profile, and finally a lower case letter that is an individual identifier.³⁰ In the other nomenclature system, the genes are designated as aac, aad, and aph followed by a capital letter that identifies the site of modification and a number to provide a unique identifier to different genes.²⁶ In addition to AMEs, methylation of 16S rRNA by ArmA enzyme, conferring high-level aminoglycoside resistance have been reported as a novel mechanism of resistance against aminoglycosides in A. baumannii, primarily in the Far East,^39,41 Europe,^16,32 and North America.⁹

The aim of the current study was to provide an insight into the frequency of genes encoding AMEs and 16S rRNA methylase, ArmA in A. baumannii isolates belonging to different sequence groups (SGs).

Materials and Methods

Bacteria

A total of 89 consecutive nonduplicate isolates of Acinetobacter spp. were collected during January through April, 2014 from different hospitals of Tabriz and Orumieh in the north west of Iran. All isolates were initially identified using conventional microbiological methods.³⁸ Later, species identification was confirmed by PCR amplification of the intrinsic bla_oxa-51-like allele³⁶ and partial sequencing of rpoB gene as described previously.¹⁸

Determination of clonal lineage

SGs or international clone (IC) of isolates were identified by trilocus sequence-based typing (3LST) using two multiplex PCR sets amplifying Group 1 (assigned as IC II), Group 2 (assigned as IC I), and Group 3 (assigned as IC III) alleles of the outer membrane protein A (ompA), chaperone–subunit usher E (csuE), and the intrinsic carbapenemase (bla_oxa-51-like) genes as described previously.³⁵

Susceptibility testing

The minimum inhibitory concentrations (MICs) of five representative aminoglycosides, including gentamicin, amikacin, kanamycin, netilmicin, and tobramycin, were determined using the E-test (Liofilchem) method and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).⁵

Detection of aminoglycoside resistance genes

PCR assays were performed to detect the presence of genes encoding the following AMEs: APH(3′)-Ia (aphA1), APH(3′)-VIa (aphA6), AAC(3′)-Ia (aacC1), AAC(3′)-IIa (aacC2), AAC(6′)-Ib (aacA4), AAC(6′)-Ih, ANT(2′)-Ia (aadB), and ANT(3′)-Ia (aadA1) with specific primers and PCR conditions as described previously.^15,25 In addition, all isolates were subjected to the separate PCR for amplification of the armA using the following primers: forward: 5′-ATT CTG CCT ATC CTA ATT GG-3′ and reverse: 5′-ACC TAT ACT TTA TCG TCG TC-3′ with identical PCR conditions as described previously.¹¹

Results

Species identification

Of 89 isolates identified as Acinetobacter spp. phenotypically, bla_oxa-51-like gene amplification and rpoB sequencing could confirm 87 isolates as A. baumannii. These isolates were collected from two main cities located in the north west of Iran: 76 A. baumannii isolates from university teaching hospitals in Tabriz and 11 isolates from Orumieh. Source of these isolates was wound (n = 40), followed by tracheal aspirates (n = 20), blood (n = 11), urine (n = 8), catheter (n = 2), cerebrospinal fluid (n = 2), and other body fluids (n = 4).

SG typing

3LST revealed the presence of nine different SGs tested among A. baumannii isolates. The majority of isolates (n = 74) belonged to defined SGs 1, 2, and 3 (corresponding to IC II, I, and III, respectively). The remaining 13 isolates belonged to 6 novel variants of SGs, defined according to the new combination of products obtained in the two separate multiplex PCRs (Table 1).

Table 1.

Novel Combinations of Sequence Groups

	Group 1 PCR			Group 2 PCR
Sequence group	csuE	ompA	bla_oxa-51like	csuE	ompA	bla_oxa-51-like
SG4	−	+	+	+	−	−
SG5	−	−	+	+	+	−
SG6	+	−	−	−	+	+
SG7	−	+	−	+	−	−
SG8	−	−	−	−	+	−
SG9	+	+	−	−	−	+

PCR, polymerase chain reaction; SG, sequence group; +, presence of gene; −, absence of gene.

Susceptibility testing results

Table 2 depicts the susceptibility of A. baumannii isolates belonging to various SGs toward five aminoglycosides and comparison of them between SG1–9. Among all tested aminoglycosides, netilmicin had the highest activity (67.8%) against isolates of all SGs. In contrast, kanamycin displayed least activity, with 5.7% of isolates observed as susceptible. Seventy-seven (88.5%) isolates showed high-level resistance (HLR) to at least one of the tested aminoglycosides with MICs ≥256 μg/ml. The HLR rate for gentamicin, amikacin, kanamycin, tobramycin, and netilmicin was 57.4%, 54%, 87.3%, 47.1%, and 21.8%, respectively.

Table 2.

MICs Distribution of Aminoglycosides in Various Sequence Groups

	Gentamicin MICs (μg/ml)						Amikacin MICs (μg/ml)						Kanamycin MICs (μg/ml)						Tobramycin MICs (μg/ml)						Netilmicin MICs (μg/ml)
Sequence group (n, %)	≤4	8	16–128	≥256	MIC₅₀	MIC₉₀	≤16	32	64–128	≥256	MIC₅₀	MIC₉₀	≤16	32	64–128	≥256	MIC₅₀	MIC₉₀	≤4	8	16–128	≥256	MIC₅₀	MIC₉₀	≤8	16	32	≥256	MIC₅₀	MIC₉₀
SG1 (34, 39)	5	3	4	22	≥256	≥256	6	2	5	21	≥256	≥256	1	—	2	31	≥256	≥256	12	—	3	19	≥256	≥256	15	—	1	18	≥256	≥256
SG2 (29, 33.3)	20	—	3	6	4	≥256	11	7	6	5	32	≥256	3	3	1	22	≥256	≥256	22	1	2	4	2	≥256	25	2	1	1	4	16
SG3 (11, 12.6)	—	—	1	10	≥256	≥256	—	1	—	10	≥256	≥256	—	—	—	11	≥256	≥256	—	—	1	10	≥256	≥256	9	2	—	—	8	16
SG4 (1, 1.1)	—	—	—	1	≥256	≥256	—	—	—	1	≥256	≥256	1	—	—	—	16	16	—	1	—	—	8	8	—	1	—	—	16	16
SG5 (2, 2.2)	—		—	2	≥256	≥256	—	—	—	2	≥256	≥256	—	—	—	2	≥256	≥256	—	—	—	2	≥256	≥256	2	—	—	—	4	8
SG6 (3, 3.4)	—	—	—	3	≥256	≥256	—	—	—	3	≥256	≥256	—	—	—	3	≥256	≥256	—	—	—	3	≥256	≥256	3	—	—	—	4	8
SG7 (3, 3.4)	1	—	—	2	≥256	≥256	—	—	2	1	64	≥256	—	—	—	3	≥256	≥256	1	—	2	—	32	32	3	—	—	—	1	2
SG8 (3, 3.4)	—	—	—	3	≥256	≥256	—	—	—	3	≥256	≥256	—	—	—	3	≥256	≥256	—	—	1	2	≥256	≥256	1	2	—	—	16	16
SG9 (1, 1.1)	—	—	—	1	≥256	≥256	—	—	—	1	≥256	≥256	—	—	—	1	≥256	≥256	—	—	—	1	≥256	≥256	1	—	—	—	8	8
Total	26	3	8	50	—	—	17	10	13	47	—	—	5	3	3	76	—	—	35	2	9	41	—	—	59	7	2	19	—	—

MIC, minimum inhibitory concentration.

Comparing different SGs for aminoglycoside resistance, SG2 isolates showed more susceptibility to all tested aminoglycosides. Twenty isolates (68.9%) of SG2 had MICs ≤4 μg/ml to gentamicin, and equivalent MICs were recorded for 5 isolates (14.7%) grouped as SG1 and 1 (33.3%) isolate of SG7.

Aminoglycoside resistance genes

To study aminoglycoside resistance mechanisms, all isolates from nine SGs were screened for the occurrence of AMEs and 16S rRNA methylase, ArmA-encoding genes.

Table 3 shows frequency of AMEs and armA in different SGs with relation to their resistance phenotype.

Table 3.

Frequency of AMEs and ArmA-Encoding Genes and Susceptibility Testing Results in Various Sequence Groups

			Gentamicin MICs (μg/ml)				Amikacin MICs (μg/ml)				Kanamycin MICs (μg/ml)				Tobramycin MICs (μg/ml)				Netilmicin MICs (μg/ml)
Sequence group	Detected gene	Isolate, n (%)	≤4	8	16–64	≥256	≤16	32	64–128	≥256	≤16	32	64–128	≥256	≤4	8	16–128	≥256	≤8	16	32–64	≥256
SG1 (n = 34)	aacA4	7 (20.5)	—	—	—	7	—	—	—	7	—	—	—	7	—	—	2	5	2	—	—	5
	aac(6)-Ih	10 (29.4)	—	—	2	8	2	1	—	7	—	—	1	9	2	—	3	5	5	—	1	4
	aacC1	12 (35.2)	1	1	1	9	1	—	1	10	1	—	—	11	3	—	1	8	5	—	1	6
	aacC2	1 (2.9)	1	—	—	—	—	—	—	1	—	—	—	1	1	—	—	—	1	—	—	—
	aadA1	9 (26.4)	1	2	2	4	1	—	3	5	—	—	—	9	5	—	—	4	5	—	1	3
	aadB	11 (32.3)	2	3	—	6	1	—	5	5	—	—	—	11	5	—	2	4	8	—	1	2
	aphA1	9 (26.4)	1	3	—	5	1	—	4	4	—	—	—	9	4	—	1	4	5	—	—	4
	aphA6	16 (47)	2	3	1	10	1	—	4	11	—	—	—	16	6	—	3	7	9	—	1	6
	armA	20 (58.8)	1	—	—	19	—	—	1	19	—	—	1	19	1	—	2	17	3	—	—	17
SG2 (n = 29)	aacA4	11 (37.9)	8	—	2	1	1	2	6	2	—	1	1	9	10	—	1	—	9	—	1	1
	aac(6)-Ih	11 (37.9)	8	—	1	2	3	2	3	3	1	2	—	8	10	—	—	1	10	—	—	1
	aacC1	11 (37.9)	8	—	—	3	2	2	3	4	2	—	—	9	9	—	—	2	9	1	—	1
	aacC2	5 (17.2)	3	—	1	1	1	1	2	1	1	—	1	3	4	—	1	—	4	1	—	—
	aadA1	9 (31)	7	—	2	—	4	2	1	2	—	2	1	6	7	1	1	—	8	—	1	—
	aadB	6 (20.6)	—	—	2	4	1	—	—	5	—	1	—	5	—	1	1	4	4	—	2	—
	aphA1	1 (3.4)	1	—	—	—	1	—	—	—	—	—	—	1	1	—	—	—	1	—	—	—
	aphA6	18 (62)	11	—	2	5	1	4	5	8	—	—	1	17	13	1	1	3	15	—	2	1
	armA	3 (10.3)	3	—	—	—	—	2	—	1	—	—	—	3	3	—	—	—	3	—	—	—
SG3 (n = 11)	aacA4	1 (9)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aac(6)-Ih	2 (18.1)	—	—	—	2	—	—	—	2	—	—	—	2	—	—	—	2	2	—	—	—
	aacC1	11 (100)	—	—	1	10	—	1	—	10	—	—	—	11	—	—	1	10	9	2	—	—
	aadA1	6 (54.5)	—	—	1	5	—	—	1	5	—	—	—	6	—	—	1	5	4	1	1	—
	aadB	10 (90.9)	—	—	—	10	—	—	—	10	—	—	—	10	—	—	—	10	8	—	2	—
	aphA1	1 (9)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aphA6	9 (81.8)	—	—	1	8	—	1	—	8	—	—	—	9	—	—	1	8	7	2	—	—
	armA	1 (9)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
SG4	aacC2	1 (100)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	—	1	—	—
SG5 (n = 2)	aacA4	1 (50)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aac(6)-Ih	1 (50)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aacC1	1 (50)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aadB	2 (100)	—	—	—	2	—	—	—	2	—	—	—	2	—	—	—	2	2	—	—	—
	aphA6	1 (50)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
SG6 (n = 3)	aacA4	1 (33.3)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aacC1	3 (100)	—	—	—	3	—	—	—	3	—	—	—	3	—	—	—	3	3	—	—	—
	aadB	3 (100)	—	—	—	3	—	—	—	3	—	—	—	3	—	—	—	3	3	—	—	—
	aphA6	3 (100)	—	—	—	3	—	—	—	3	—	—	—	3	—	—	—	3	3	—	—	—
SG7 (n = 3)	aac(6)-Ih	2 (66.6)	—	—	—	2	—	—	1	1	—	—	—	2	—	—	2	—	2	—	—	—
	aadB	3 (100)	—	—	1	2	—	1	1	1	—	—	—	3	—	—	3	—	3	—	—	—
	aphA1	2 (66.6)	—	—	—	2	—	—	1	1	—	—	—	2	—	—	2	—	2	—	—	—
	aphA6	3 (100)	1	—	—	2	—	—	2	1	—	—	—	3	1	—	2	—	3	—	—	—
SG8 (n = 3)	aacA4	1 (33.3)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	1	—	—	1	—	—
	aacC1	2 (66.6)	—	—	—	2	—	—	—	2	—	—	—	2	—	—	1	1	—	2	—	—
	aadB	3 (100)	—	—	—	3	—	—	—	3	—	—	—	3	—	—	1	2	1	2	—	—
	aphA1	1 (33.3)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	1	—	—	1	—	—
	aphA6	3 (100)	—	—	—	3	—	—	—	3	—	—	—	3	—	—	1	2	1	2	—	—
SG9 (n = 1)	aacA4	1 (100)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aadB	1 (100)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—
	aphA6	1 (100)	—	—	—	1	—	—	—	1	—	—	—	1	—	—	—	1	1	—	—	—

AME, aminoglycoside-modifying enzyme.

Among 84 isolates found resistant to at least one of the eight studied aminoglycosides, 14 (16.6%) harbored one AME gene, 19 (22.6%) isolates had two AME genes, 18 (21.4%) isolates possessed three AME genes, 23 (27.3%) isolates had four genes, and 6 (7.1%) isolates disclosed five genes. Four (4.7%) isolates were negative for any type of the studied AME genes. Among eight investigated AME-encoding genes, aphA6 was the predominant gene with a positive rate of 47%, 62%, 81.8%, and 50% for SG1, SG2, SG3, and SG5, respectively. Regarding remaining SGs, all isolates of SGs 6–9 were observed positive for aphA6.

When A. baumannii isolates were screened for the presence of armA by PCR, 58.8%, 10.3%, and 9% isolates of SG1, SG2, and SG3 groups, respectively were positive for this gene. Isolates of other SGs were negative for this gene.

Correlation analysis between aminoglycoside resistance and investigated genes

Statistical analysis of various genes responsible for resistance to aminoglycosides in clinical isolates of A. baumannii from different SGs revealed that in SG1, resistance to amikacin and netilmicin were significantly associated with armA (p < 0.05). Regarding tobramycin resistance, presence of armA and aacA4 were correlated with resistance phenotype. However, there was no significant association between the presence of investigated genes and resistance to gentamicin or kanamycin in SG1. In contrast, among SG2 isolates, resistance to these aminoglycosides was found to be associated with the existence of aadB and aphA6, respectively. Other observed significant correlations were amikacin resistance with positive results of aphA6 and tobramycin resistance with the presence of aadB in SG2 isolates.

Discussion

A. baumannii has established itself as a successful pathogen. It is responsible for numerous nosocomial infections, which are caused mostly by outbreak strains.²⁷ From mid 1990s, most outbreak strains from worldwide hospitals were classified into epidemic clones, so called the International clones (IC) I, II, and III with a marked predominance of IC II.^6,8,37

In the present study, 87 A. baumannii isolates were grouped into 9 different SG types (SGs 1–3 consistent with IC II, I, III, respectively), and majority (72.4%) of these belonged to 2 multinational lineages, IC I (33.3%) and IC II (39%). Although only limited information is available concerning A. baumannii isolates in Iran, predominant circulation of IC II has been reported in two previous studies from Iran.^14,28 However, according to the published data by Bahador et al.² on 91 A. baumannii isolates from patients in intensive care units of three hospitals in Iran, the novel variant SG7, has been introduced as a pan-Iranian clone, which is in contrast to our results. This disparity may be explained in terms of selection of patient groups.

3LST in our study did not assign 13 (14.9%) isolates to any of the 3 major SGs. Among these novel variants that were assigned as SG4–9, SG6, and SG7 have been previously reported by Towner et al.³⁴ among carbapenem-resistant A. baumannii spreading throughout Europe. Diversity of the ompA, csuE, and bla_oxa-51-like genes in A. baumannii has been reported in some studies around the world.^2,13,14,34 It is suggestive to determine the SGs of sporadic isolates as well as their characterization, as it may enable new insights into the reasons for successful dissemination and/or pathogenicity.

Carbapenem resistance and associated mechanisms in A. baumannii isolates from different geographic regions were investigated.^17,21,28,29 However, there is paucity of literature for aminoglycoside resistance in different SGs of A. baumannii.

The results of antimicrobial susceptibility testing represented netilmicin as the most active aminoglycoside against A. baumannii isolates in our study, with susceptibility rate of 67.8% and MIC₅₀ ≤8 μg/ml. This finding is in agreement with other investigators^4,33 and was predictable because of limited prescription of this antibiotic in clinics. Conversely, 80% of our isolates showed MIC ≥16 μg/ml toward amikacin, which is higher than the results previously reported from Iran,¹⁰ Spain,³¹ Italy,³¹ and United States,²² whereby 38.2%, 37%, 37.8%, and 47% isolates were observed to be amikacin resistant, respectively. Amikacin and tobramycin are the therapeutic options for infections caused by multidrug-resistant Acinetobacter isolates, usually being used in conjunction with another active antimicrobial agent.²⁰ Thus, this high frequency of amikacin-resistant A. baumannii strains could anticipate future therapeutic problems for A. baumannii infections.

Considering the clonality of isolates, our observation demonstrated the least rate of resistance to tested aminoglycosides among isolates belonging to SG2 (IC I). This finding is surprising and has not been reported yet and requires further investigation with larger number of A. baumannii isolates to support our finding.

Based on the molecular analysis of aminoglycoside-resistant strains, AMEs have been suggested as the main mechanism associated with aminoglycoside resistance.³⁹ A wide array of AMEs have been reported in A. baumannii.^1,14,24 In the present study, isolates of SG1 (IC II) and SG2 (IC I) displayed the most variable combinations of AMEs (23 and 21, respectively). The diversity of aminoglycoside resistance genes in IC I and IC II have earlier been reported in a set of strains from the Czech Republic and other European countries and have supported this view that these clones are relatively old groups that have been undergoing diversification.²⁴ In contrast to SG1 and SG2, lineage SG3 in our study represents less variability in the content of AME genes with five different resistance gene profiles, which suggest their expansion from a common ancestor. The combination of aphA6-aadB-aacC1-aadA1 was the most detected combination accounting SG3 revealed in 6 (54.5%) isolates, and all of the isolates with this combination were found to be resistant to gentamicin, amikacin, kanamycin, and tobramycin.

In our study, aphA6 was the predominant gene present in SG1 (47%) and SG2 (62%) groups, in addition to SG6, SG7, SG8, and SG9 with a detection rate of 100%. Regarding clonal lineage SG3, AAC(3′)-Ia and ANT(2′)-Ia were the dominant AMEs, detected in 100% and 90.9% isolates, respectively. These findings strongly differed from those reported earlier by Nemec et al.,²⁴ whereby AAC(3′)-Ia, ANT(3′)-Ia and APH(3′)-Ia were the most detected AMEs in both IC I and IC II. In addition, comparing our results with their study, which documented ANT(2′)-Ia and APH(3′)-VIa as only detected AMEs in the international clonal lineage III, and considering the isolation period of their strains (1997–1998), it could be related to the evolution of aminoglycoside resistance pools in this clonal lineage.

In recent years, the production of 16S rRNA methylase by gene armA has also been implicated in aminoglycoside resistance, which usually confer HLR to 4,6-substituted deoxystreptamines, including arbekacin, amikacin, kanamycin, tobramycin, and gentamicin, by impeding their access to the site of action.⁹ ArmA-producing A. baumannii strains have been reported from many countries.^9,19,40 The overall carriage of armA (27.5%) in our isolates was higher than that reported in a recent study from Iran, in which merely 10.6% of A. baumannii isolates had armA.¹ However, in accordance with the abovementioned study, which found three armA-positive A. baumannii susceptible to gentamicin or tobramycin, we found three of the SG2 isolates to harbor armA, which displayed susceptibility to gentamicin, tobramycin, and netilmicin, as well as intermediate resistance to amikacin. In addition, among our SG1 isolates, susceptibility to gentamicin and/or tobramycin as well as netilmicin was seen in one and three armA-positive isolates, respectively. Although susceptibility to netilmicin was expected as action of this aminoglycoside was not affected by ArmA, susceptibility to gentamicin and tobramycin in armA-containing isolates could be explained by mutation in the active site sequence of the enzyme and/or defective expression of this 16S rRNA methylase.

In the present study, we found one isolate belonging to SG1, negative for the presence of all eight tested AMEs or armA, but had resistance to amikacin (MIC ≥256 μg/ml), kanamycin (MIC ≥256 μg/ml), and gentamicin (MIC = 64 μg/ml). This finding implies the role of other resistance mechanisms against aminoglycosides such as impermeability of the drugs, efflux pumps, or other types of 16S rRNA methylase.¹²

In conclusion, our results revealed a definite relation between the genotypes being present in various clonal lineages in the north west of Iran, which were noticed for the first time. We could find three clonal lineages analogous to international clones I, II, and III and other novel SGs were noticed. The correlation between aminoglycoside resistances and the presence of AMEs within these SGs groups is another finding which is of notable concern.

Footnotes

Acknowledgments

The authors thank Ms. Leila Dehghani and Ms. Zeila KhajeMohammadi for their technical assistance in the collection of clinical isolates and phenotypic workup. This work was supported by the Immunology Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 93/32). This article is part of the PhD thesis of the corresponding author registered in the Tabriz University of Medical Sciences.

Disclosure Statement

No competing financial interests exist.

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