Genotyping and molecular characterization of clinical Acinetobacter baumannii isolates from a single hospital in Southwestern Iran

ABSTRACT Acinetobacter baumannii

(A. baumannii) is a pathogen responsible for nosocomial infections among the hospitalized patients. The aim of this study was to investigate genotyping and molecular characterization and to examine the biofilm formation ability of A. baumannii isolates. In total, 70 A. baumannii isolates were collected from patients admitted to Imam Khomeini Hospital in Ahvaz, Southwestern Iran. Minimum inhibitory concentrations (MIC) test was performed using Vitek 2 system. The presence of genes encoding metallo-β-lactamases, oxacillinases, and integrase and the biofilm formation ability were then evaluated. Multiple locus variable-number tandem repeat (VNTR) analysis (MLVA) typing and multiplex PCR were performed to determine the genetic relationships. The bla_OXA-23-like gene had the highest prevalence. The frequency of genes encoding bla_SPM, bla_IMP, and bla_VIM among MDR A. baumannii isolates were 12 (17.1%), 18 (25.7%), and 22 (31.4%), respectively. Moreover, 46 isolates (75.4%) harbored class I integron and 10 isolates (16.39%) carried class II integron. The number of weak, moderate and strong biofilm-producing isolates were 3 (4.3%), 7 (10%), and 55 (78.5%), respectively. The results showed that 70 A. baumannii isolates were grouped into 12 distinct MLVA types with five clusters and four singleton genotypes. In addition, 25 (35.7%) isolates were assigned to international clone (IC) variants, 37 (52.8%) isolates belonged to group 1 (IC II), and 8  (11.4%) isolates belonged to group 2 (IC I). Our findings revealed that the population structure of the A. baumannii isolates was genetically diverse. More focus on genetic variation and antibiotic resistance of A. baumannii isolates are recommended.

Introduction

Acinetobacter baumannii is a ubiquitous bacterium known to be implicated in nosocomial infections, particularly in hospitalized patients. Infection caused by this hospital-associated pathogen often leads to significant morbidity and mortality [1]. Clinical isolates of A. baumannii have the ability to remain in hospital environments, persistently, for a prolonged period of time, commonly giving rise to hospital outbreaks [2].

In previous decades, A. baumannii resistance to various antimicrobial drugs has markedly escalated. This pattern of antibiotic resistance frequently varies in time and from one area to another or even within the same area [3,4]. Recently, multidrug-resistant (MDR) A. baumannii strains have emerged as formidable nosocomial pathogens. In many developing countries, doctors face serious problems in the management of patients infected with MDR A. baumannii, imposing considerable challenges on healthcare systems owing to treatment failure and increased mortality [5,6]. Carbapenem antibiotics are the most appropriate drugs of choice for treating infections caused by MDR A. baumannii strains. Overuse of broad-spectrum carbapenems and/or cephalosporins is a major risk factor for colonization or infection with carbapenemase-producing A. baumannii strains. Unfortunately, the prevalence of carbapenem resistance in the aforementioned strains is increasingly growing [7].

The most significant mechanisms of resistance to antibiotics include target protection or modification, antibiotic enzymatic degradation, decreased permeability, and drug extrusion by the efflux pump. Resistance to carbapenems in A. baumannii is related to a variety of enzymes, including metallo-β-lactamases (IMP, VIM, and NDM) and OXA-type carbapenemases (OXA-24, OXA-58, OXA-51, and OXA-23) [8,9]. MDR A. baumannii infections are typically associated with high mortality, and patients acquire infection from other patients and from different environmental sources [10]. Some clinical isolates of A. baumannii have a high propensity for biofilm formation and occasionally cause medical device-related infections. Biofilm formation is an important pathogenic mechanism in such infections and has a key role in the long-term survival of bacteria under environmental pressures [11].

Based on epidemiological studies and population genetic investigations of A. baumannii, there are several different typing methods, including multiple-locus variable number tandem repeats (VNTRs) analysis (MLVA), pulsed-field gel electrophoresis (PFGE), whole genome sequencing (WGS), multilocus sequence typing (MLST), and PCR-based sequence group (SG) profiling [12–14]. This study aimed to investigate the presence of genes encoding metallo-β-lactamases, oxacillinases, integrase and to scrutinize the biofilm formation ability of A. baumannii isolates. MLVA typing and multiplex PCR were used to determine the genetic relationships of the isolates collected from patients admitted to Imam Khomeini Hospital in Ahvaz, Southwestern Iran.

Materials and methods

Bacterial isolates and identification

This cross-sectional study was confirmed by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (no. IR.AJUMS.REC.1397.793) in accordance with the Declaration of Helsinki and conducted between October 2018 and July 2019. Before study initiation, written informed consents were provided by all the patients. A total of 70 non-repetitive A. baumannii isolates were collected from clinical samples (tracheal aspirates, blood, cerebrospinal fluid [CSF] urine, catheter, and pleural fluid) of patients who were referred to Imam Khomeini Hospital in Ahvaz. The isolates were initially characterized by standard tests, and their final identification was performed by PCR of bla_OXA-51-like gene and multiplex of gyrB. The A. baumannii ATCC19606 was selected as a positive control [15,16].

Antimicrobial susceptibility testing

The minimum inhibitory concentrations (MICs) of 17 antibiotics (ampicillin-sulbactam, piperacillin, cefepime, ceftazidime, cefotaxime, amikacin, meropenem, imipenem, ciprofloxacin, levofloxacin, minocycline, gentamicin, tobramycin, tetracycline, colistin, tigecycline, and trimethoprim-sulfamethoxazole) against A. baumannii isolates were determined using Vitek 2 system (bioMérieux, Marcy l’Etoile, France). The MICs of all the antibiotics were interpreted by the mentioned system according to the CLSI guidelines [17]. The MIC₅₀ and MIC₉₀ of each antibiotic were then calculated.

The MIC breakpoints of resistance were ≥ 4 µg/ml for colistin and ≥ 8 µg/ml for imipenem and meropenem. For tigecycline, no MIC interpretive breakpoint was recommended by the EUCAST and CLSI. MIC ≥ 8 μg/ml was proposed as the resistant breakpoint according to the criteria suggested by Jones et al. [18]. MDR Acinetobacter isolates are defined as strains resistant to at least three classes of antimicrobial agents, including all penicillins and cephalosporins, aminoglycosides, and fluoroquinolones [19]. Quality control for the MIC analysis was performed with Escherichia coli ATCC 25,922 and Pseudomonas aeruginosa ATCC 27,853, and the phenotypic detection of MBL production was accomplished as per a former study [20].

PCR amplification of the β-lactamases-encoding genes and identification of integrons

Detection of genes encoding oxacillinases (bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like, bla_OXA-58-like, and bla_OXA-143-like) [21] and bla genes (bla_SIM, bla_NDM, bla_VIM, bla_IMP, and bla_SPM) was conducted by using the specific primers described previously [22,23]. Bacterial DNA was obtained by the boiling method [23].

PCR was performed for each pair of primers in a total volume of 25 µl in a reaction tube containing 2 µl of DNA template, 10 µl of Amplicon Master Mix, 1 µl of each forward and reverse primer (2.5 pmol), and 11 µl of distilled water. PCR reactions included 30 cycles of amplification in a Mastercycler (Eppendorf, Germany) under the following conditions: denaturation at 95 °C for five min, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 6 min. Amplified products were visualized following electrophoresis on a 1% agarose gel stained with safe stain in a Tris-Borate-EDTA (TBE) buffer (Promega, USA). PCR was also performed to detect the presence of class I, II, and III integrons by the amplification of integrase genes, including intI1-, intI2-, and intI3-specific primers [24].

In vitro bacterial biofilm assay

The biofilm formation ability of the A. baumannii isolates on 96-well polystyrene microtiter plates (MTP) was assessed using the crystal violet staining (CVS) method, as formerly described [25]. For evaluating biofilm formation, Muller Hinton Broth and A. baumannii ATCC19606 were used as negative and positive controls, respectively. The results were interpreted according to the criteria described by Zhang et al. [26]. The isolates were strong biofilm producers when 4 × ODc < OD₅₇₀, moderate biofilm producers when 2 × ODc < OD₅₇₀ ≤ 4 × ODc, weak biofilm producers when ODc < OD₅₇₀ ≤ 2 × ODc, and no biofilm producers when OD₅₇₀ ≤ ODc. All the experiments were conducted in triplicate.

Molecular typing methods

For molecular typing, two multiplex PCRs were performed to selectively amplify the alleles of chaperone-subunit usher E (csuE), the outer membrane protein A (ompA), and the intrinsic carbapenemase (bla_OXA-51-like) gene, and to determine the sequence groups and the corresponding major international clones (ICs) I–III according to Turton et al. [27]. The amplification reaction was carried out by using a thermal cycler (Mastercycler Eppendorf, Germany) with an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 1 min, and a final extension step was conducted at 72 °C for 5 min. The MLVA typing of A. baumannii isolates was carried out using the MLVA-8 scheme method designed by Pourcel et al. [13]. The MLVA-8 scheme profiles in each isolate were identified by the number of repeats estimated at each VNTR locus.

Statistical analysis

Descriptive data were analyzed using Microsoft excel and SPSS version 22 statistics software (IBM Corporation, Armonk, NY, USA). Fisher’s exact test was applied to analyze intergroup significance. In addition, p < 0.05 was considered as statistical significance level. The results are presented as descriptive statistics in terms of relative frequency.

Results

Studied population, sampling, and antibiotic susceptibility

In the current study, 70 non-duplicative A. baumannii isolates were collected from 42 (60%) males and 28 (40%) females with the mean age of 42.5 ± 10 (ranged 5–80) years, with the maximum number of cases in the age group of 41 to 60 years (n = 36). These isolates were obtained from different clinical specimens, including tracheal aspirates (26; 37.1%), blood (9; 12.8%), CSF (5; 7.1%), urine (8; 11.4%), catheter (15; 21.4%), and pleural fluid (7; 10%). The frequencies of the A. baumannii isolates collected from different wards were 31 (44.2%) in the intensive care unit (ICU), as well as 10 (14.3%) in surgery, 8 (11.5%) in urology, 6 (8.6%) in infectious diseases, 7 (10%) in general, 5 (7.1%) in pediatric, and 3 (4.3%) in neurology units (Table 1).

Table 1.

Clinical data of the patients with A. baumannii isolates. ID: infectious diseases; ICU: intensive care unit; M: male; F: female; TA: tracheal aspirates; CSF: Cerebrospinal fluid; PF: pleural fluid.

Strain ID	Ward	Gender	Clinical samples	Date of collection
Ab 58	General	F	TA	May 2018
Ab 59	Surgery	M	PF	May 2018
Ab 66	ICU	F	CSF	June 2019
Ab 43	Neurology	F	Catheter	February 2019
Ab 45	Urology	M	Blood	February 2019
Ab 46	General	M	TA	March 2019
Ab 48	Surgery	F	TA	March 2019
Ab 49	ID	F	TA	March 2019
Ab 50	ICU	M	Catheter	March 2019
Ab 51	Pediatric	M	TA	March 2019
Ab 52	Urology	F	Urine	April 2019
Ab 53	ICU	M	Blood	April 2019
Ab 54	ICU	M	Catheter	April 2019
Ab 60	ID	M	Catheter	May 2018
Ab 61	ICU	F	TA	May 2018
Ab62	ICU	F	TA	May 2018
Ab 63	ICU	M	Blood	June 2019
Ab 64	General	M	Catheter	June 2019
Ab 67	ID	M	Urine	June 2019
Ab 68	ICU	M	PF	June 2019
Ab 69	ICU	F	CSF	June 2019
Ab 70	Urology	F	Catheter	June 2019
Ab 65	Surgery	M	Catheter	June 2019
Ab 15	ICU	F	Catheter	November 2018
Ab 16	Pediatric	M	Blood	December 2018
Ab 17	ICU	F	TA	December 2018
Ab 18	ICU	M	Catheter	December 2018
Ab 19	Urology	F	TA	December 2018
Ab 20	ID	F	PF	December 2018
Ab 21	ICU	M	Blood	December 2018
Ab 22	Surgery	M	TA	December 2018
Ab 23	Neurology	F	Catheter	December 2018
Ab 24	General	M	TA	December 2018
Ab 25	ICU	M	Catheter	December 2018
Ab 26	ID	F	Blood	December 2018
Ab 27	ICU	M	TA	December 2018
Ab 28	Surgery	M	Blood	December 2018
Ab 1	ICU	M	Urine	October 2018
Ab 2	ICU	F	PF	October 2018
Ab 3	Urology	M	Blood	October 2018
Ab 4	ICU	F	Catheter	October 2018
Ab 7	General	M	Blood	October 2018
Ab 5	Pediatric	F	TA	October 2018
Ab 8	ICU	M	TA	October 2018
Ab 6	ICU	M	CSF	October 2018
Ab 10	Surgery	M	PF	October 2018
Ab 11	Neurology	M	CSF	November 2018
Ab 12	ICU	F	Catheter	November 2018
Ab 13	Surgery	M	Urine	November 2018
Ab 14	General	M	TA	November 2018
Ab 9	Urology	F	Urine	October 2018
Ab 44	ICU	M	TA	February 2019
Ab 47	ICU	M	Urine	March 2019
Ab 55	Urology	M	TA	April 2019
Ab 29	ID	F	Catheter	January 2019
Ab 30	Pediatric	M	TA	January 2019
Ab 31	ICU	M	PF	January 2019
Ab 32	Urology	F	TA	January 2019
Ab 33	ICU	M	TA	January 2019
Ab 34	Surgery	F	Urine	January 2019
Ab 35	ICU	F	Catheter	January 2019
Ab 36	General	M	TA	January 2019
Ab 37	Pediatric	M	TA	January 2019
Ab 38	Surgery	M	Urine	February 2019
Ab 39	ICU	M	CSF	February 2019
Ab 40	ICU	F	TA	February 2019
Ab 41	Surgery	F	TA	February 2019
Ab 42	ICU	M	PF	February 2019
Ab 56	ICU	F	TA	May 2018
Ab 57	ICU	M	TA	May 2018

According to antibiotic susceptibility testing, among 70 A. baumannii isolates screened, 61 (87.1%) were MDR and 9 (12.8%) were extensive drug-resistant (XDR) isolates. The results showed 100% resistance to ceftazidime, cefepime, and cefotaxime, indicating no effect on A. baumannii isolates, while colistin and tigecycline were the most active antimicrobial agents (with 2.8% and 45.7% resistance, respectively). Besides, among A. baumannii isolates, resistance to other antibiotics was ≥ 50% (Table 2).

Table 2.

Antibiotic susceptibility pattern of A. baumannii isolates.

Antibiotic		GEN	TOB	LEV	TET	MIN	CTZ	CFP	PIP	TGC	AMP/S	COL	CTX	SXT	AMK	CIP	IMI	MER
Susceptibility pattern	RN (%)	58 (82.8)	52 (74.3)	65 (92.8)	54 (77.1)	43 (61.4)	70 (100)	70 (100)	68 (97.1)	32 (45.7)	55 (78.6)	2 (2.8)	70 (100)	67 (95.7)	59 (84.2)	65 (92.8)	50 (71.4)	52 (74.2)
	IN (%)	2 (2.8)	4 (5.7)	0 (0.0)	0 (0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	2 (2.8)	0(0.0)	3 (4.3)	0 (0.0)
	SN (%)	10 (14.3)	14 (20)	5 (7.1)	16 (22.8)	27 (38.5)	0(0.0)	0(0.0)	2(2.8)	38 (54.3)	15(21.4)	68 (97.1)	0(0.0)	3 (4.3)	9 (12.8)	5 (7.1)	17 (24.2)	18 (25.7)
Interpretive categories and MIC breakpoints (µg/ml)	R	≥16	≥16	≥8	≥16	≥16	≥32	≥32	≥128	≥8	≥32/16	≥4	≥64	≥4/76	64	≥4	≥8	≥8
	I	8	8	4	8	8	16	16	32–64	-	16/8	-	16–32	-	32	2	4	4
	S	≤4	≤4	≤2	≤4	≤4	≤8	≤8	≤16	<8	≤8/4	≤2	≤8	≤ 2/38	16	≤1	≤2	≤2
	Range	2 to ≥240	1 to ≥240	1 to ≥ 240	2 to 256	2 to 256	4 to ≥ 256	4 to ≥ 256	5 to ≥ 240	0.25 to 64	2/1 to ≥ 256/128	0.125 to 32	4 to ≥ 256	5 to 240	8 to ≥ 256	0.1 to ≥ 240	0.25 to 256	0.25 to 256

GEN: gentamicin; TOB: tobramycin; LEV: levofloxacin; TET: tetracycline; MIN: minocycline; CTZ: ceftazidime; CFP: cefepime; PIP: piperacillin; TGC: tigecycline; Amp/S: ampicillin/Sulbactam; COL: colistin; CTX: cefotaxime; SXT: trimethoprim-sulphamethoxazole; AMK: amikacin; CIP: ciprofloxacin; IMI: imipenem; MEM: meropenem; R: resistant; I: intermaediate; S: susceptible.

All XDR isolates had bla_OXA-23-like gene, and all belonged to clone 1, 2 or 10. Two A. baumannii isolates were resistant to colistin (MIC = 32 and 8 µg/mL), had strong biofilm formation, were XDR phenotype and belonged to ICs 1 and 2. 41 (58.5%) isolates were simultaneously resistant to imipenem and meropenem, and 52 (74.2%) out of 70 A. baumannii isolates were carbapenem-resistant (CRAB). All meropenem- and imipenem-resistant isolates contained at least one bla_OXA-24-like and/or bla_OXA-23-like carbapenemase. Among the meropenem-sensitive and -resistant isolates, 18 (25.7%) and 52 (74.2%) isolates had MICs ranging from 0.25 to ≤ 2 and from ≥ 8 to 256 µg/ml, respectively. The MICs for imipenem ranged from 0.25 to ≤ 2 µg/mL in 17 (24.2) isolates, while 50 (71.4%) isolates had MICs ≥ 8–256 µg/ml for this antibiotic. Three isolates also showed MICs in the intermediate range (4 µg/ml) for imipenem (Table 2).

Frequency of β-lactamases-encoding genes and characterization of integrons

PCR results displayed the presence of bla_OXA-51-like gene in all the isolates, whereas no isolate harbored bla_OXA-143-like gene. The bla_OXA-23-like gene had the highest frequency (46 isolates, 65.7%). The combination of bla_OXA-23-like/bla_OXA-24-like and bla_OXA-23-like/bla_OXA-24-like/bla_OXA-58-like genes were found in 8 (11.4%) and 3 (4.2%) isolates, respectively. The coexistence of the bla_OXA-23-like and bla_OXA-58-like genes occurred only in 6 isolates (8.5%).

The frequency rates of the genes encoding bla_SPM, bla_IMP, and bla_VIM among A. baumannii isolates were 12 (17.1%), 18 (25.7%), and 22 (31.4%), respectively. The bla_VIM gene coexisted with bla_IMP in 9 (12.8%) isolates; however, bla_NDM and bla_SIM were not identified in these isolates. Among CRAB isolates, 41 were phenotypically recognized as MBL-producing isolates, of which 8, 16, and 11 strains were positive for the bla_SPM, bla_VIM, and bla_IMP genes, and no other MBLs were detected. Also, in the remaining 6 (12.8%) isolates, the bla_VIM gene coexisted with bla_IMP.

PCR amplification of integron genes showed that 46 isolates (75.4%) harbored class I integron, and 10 isolates (16.39%) carried class II integron. Class III integron was not identified in any isolates. Meanwhile, 5 isolates (8.19%) had both classes I and II integrons, and 9 isolates did not have these integron genes. Antibiotic susceptibility patterns of integron-positive and -negative isolates are shown in Table 3. All integron-positive isolates were resistant to cephalosporins, and the most effective antibiotic against these isolates was colistin. No significant correlation was observed between the presence of integrons and antibiotic resistance frequency, except for tetracycline with p = 0.025.

Table 3.

Relationship between antibiotic susceptibility pattern with integron and biofilm formation in A.baumannii isolates.

Antibiotic		Integron-positive isolatesN (%)				Integron-negative isolates N (%)				p value		Strong biofilm-producing isolates N (%)				Biofilm-negative isolates N (%)				p value
		R	I	S		R	I	S				R	I	S		R	I	S
Tigecycline		29 (47.5)	0 (0)	32 (52.45)		3 (33.3)	0 (0)	6 (66.6)		0.494		25 (45.45)	0 (0)	30 (54.54)		1(20)	0 (0)	4 (80)		0.377
Colistin		2 (3.27)	0 (0)	59 (96.7)		0 (0)	0 (0)	9 (100)		1		2 (3.63)	0 (0)	53 (96.36)		0 (0)	0 (0)	5 (100)		1
Meropenem		45(73.77)	0 (0)	16 (26.22)		7 (77.7)	0 (0)	2 (22.2)		1		40 (72.7)	0 (0)	15 (27.27)		5 (100)	0 (0)	0 (0)		0.318
Imipenem		43(70.5)	3 (4.9)	15 (24.59)		7(77.7)	0 (0)	2(22.2)		1		39 (70.9)	3 (5.45)	13 (23.6)		4 (80)	0 (0)	1 (20)		1
Ampicillin-sulbactam		47 (77)	0 (0)	14 (22.9)		8 (88.8)	0 (0)	1 (11.1)		0.672		41 (74.5)	0 (0)	14 (25.45)		5 (100)	0 (0)	0 (0)		1
Tetracycline		50 (81.9)	0 (0)	11 (18)		4 (44.4)	0 (0)	5 (55.5)		0.025		46 (83.6)	0 (0)	9 (16.36)		3 (60)	0 (0)	2 (20)		0.224
Minocycline		40 (65.57)	0 (0)	21 (34.4)		3(33.3)	0 (0)	6 (66.6)		0.079		37 (67.27)	0 (0)	18 (32.7)		2 (40)	0 (0)	3 (60)		0.337
Ceftazidime		61 (100)	0 (0)	0 (0)		9 (100)	0 (0)	0 (0)		-		55 (100)	0 (0)	0 (0)		5 (100)	0 (0)	0 (0)		-
Cefepime		61(100)	0 (0)	0 (0)		9 (100)	0 (0)	0 (0)		-		55 (100)	0 (0)	0 (0)		5 (100)	0 (0)	0 (0)		-
Piperacillin		59 (96.7)	0 (0)	2 (3.27)		9 (100)	0 (0)	0 (0)		1		54 (98.1)	0 (0)	1 (1.8)		4 (80)	0 (0)	1 (20)		0.169
Cefotaxime		61(100)	0 (0)	0 (0)		9 (100)	0 (0)	0 (0)		-		55 (100)	0 (0)	0 (0)		5 (100)	0 (0)	0 (0)		-
Trimethoprim-sulfamethoxazole		58 (95)	0 (0)	3 (5)		9 (100)	0 (0)	0 (0)		1		53 (96.36)	0 (0)	2 (3.6)		5 (100)	0 (0)	0 (0)		1
Amikacin		52 (85.2)	2 (3.27)	6 (9.8)		7 (77.7)	0 (0)	2 (22.2)		0.465		45 (81.8)	2 (3.6)	8 (14.5)		4 (80)	0 (0)	1 (20)		1
Ciprofloxacin		57 (93.4)	0 (0)	4 (6.55)		8 (88.8)	0 (0)	1 (11.1)		0.508		50 (90.9)	0 (0)	5 (9.09)		5 (100)	0 (0)	0 (0)		1
Gentamicin		49 (80.3)	2 (3.27)	10 (16.39)		9 (100)	0 (0)	0 (0)		0.497		46 (83.6)	1 (1.8)	8 (14.5)		4 (80)	0 (0)	1 (20)		0.612
Tobramycin		44 (72.1)	3 (4.9)	14 (22.9)		8 (88.8)	1 (11.1)	0 (0)		0.193		41 (74.5)	2 (3.6)	12 (21.8)		3 (60)	1 (20)	1 (20)		0.289
Levofloxacin		56 (91.8)	0 (0)	5 (8.19)		9 (100)	0 (0)	0 (0)		1		50 (90.9)	0 (0)	5 (9.09)		5 (100)	0 (0)	0 (0)		1

R: resistant; I: intermaediate; S: susceptible

Biofilm formation assay

Evaluation of biofilm formation was performed using the MTP method. Among all the isolates, 5 (7.1%) were non-biofilm producers, while the numbers of weak, moderate and strong biofilm producers were 3 (4.3%), 7 (10%), and 55 (78.5%), respectively. The OD₅₇₀ values for positive and negative controls were 0.411 ± 0.041 and 0.071 ± 0.010, respectively. The proportion of strong biofilm producers in various wards was 30 (96.8%) in ICU, as well as 6 (10.9%) in surgery, 5 (9%) in urology, 6 (11%) in infectious diseases, 4 (7.2%) in general, 2 (3.6%) in pediatric, and 2 (3.6%) in neurology units. Additionally, of 55 strong biofilm producers, 47 (85.45%) and 8 (14.54%) were MDR and XDR, respectively. All the weak and moderate biofilm producers were MDR phenotypes. Among strong biofilm-producing isolates, 20 and 14 were from tracheal aspirates and catheter specimens, respectively, and resistance to the entire antibiotic, except for tigecycline, colistin, and minocycline, was above 70% and was not statistically significant when compared to the biofilm-negative isolates (Table 3).

Molecular typing

The results of two multiplex PCRs indicated six different PCR-based groups (G1, G2, G4, G7, G10, and G15) among A. baumannii isolates. 37 (52.8%) and 8 (11.4%) isolates belonged to groups 1 (IC II) and 2 (IC I), respectively. Other isolates belonged to 4 IC variants PCR-based groups, including 11 (15.7%), 6 (8.5%), 5 (7.1%), and 3 (4.2%), and to G10, G4, G15 and G7, respectively. The frequency of antibiotic resistance in three epidemic lineages is shown in Table 4. Resistance to carbapenems, tetracycline, tobramycin, and levofloxacin in IC II isolates was higher than the IC I.

Table 4.

Frequency of antibiotic resistance in three epidemic lineages resistant A. baumannii isolates.

	Antibiotic resistance No(%)
IC (No.)	IMP	MER	CIP	AMK	SXT	CFT	COL	AMP/S	TGC	PIP	CFP	CFZ	TET	MIN	LEV	TOB	GEN
IC I (8)	4 (50)	5 (62.5)	8 (100)	8 (100)	8 (100)	8 (100)	1 (12.5)	8 (100)	4 (50)	8 (100)	8 (100)	8 (100)	5 (62.5)	4 (50)	7 (87.5)	5 (62.5)	7 (87.5)
IC II (37)	26 (70.2)	29 (78.3)	34 (91.8)	30 (81)	37 (100)	37 (100)	1 (2.7)	25 (67.5)	16 (43.2)	37 (100)	37 (100)	37 (100)	30 (81)	28 (75.6)	34 (91.8)	25 (67.5)	30 (81)
IC 4 variants (25)	20 (80)	18 (72)	23 (92)	21 (84)	22 (88)	25 (100)	0 (0)	22 (88)	12 (48)	23 (92)	25 (100)	25 (100)	19 (76)	11 (44)	24 (96)	22 (88)	21 (84)
Total (70)	50 (71.4)	52 (74.2)	65 (92.8)	59 (84.2)	67 (95.7)	70 (100)	2 (2.8)	55 (78.6)	32 (45.7)	68 (97.1)	70 (100)	70 (100)	54 (77.1)	43 (61.4)	65 (92.8)	52 (74.3)	58 (82.8)

GEN: gentamicin; TOB: tobramycin; LEV: levofloxacin; TET: tetracycline; MIN: minocycline; CTZ: ceftazidime; CFP: cefepime; PIP: piperacillin; TGC: tigecycline; Amp/S: ampicillin/Sulbactam; COL: colistin; CTX: cefotaxime; SXT: trimethoprim-sulphamethoxazole; AMK: amikacin; CIP: ciprofloxacin; IMI: imipenem; MEM: meropenem; R: resistant; I: intermaediate; S: susceptible; International clones: IC

Of 37 isolates belonging to IC II, 34 were biofilm producers; in addition, 17 (45.9%) were obtained from the ICU. Moreover, 4 isolates had bla_OXA-23-like/bla_OXA-24-like genes, 3 isolates possessed bla_OXA-23-like/bla_OXA-58-like genes, and 2 isolates had bla_OXA-23-like/bla_OXA-24-like/bla_OXA-58-like genes. Among 37 isolates, 33 (89.2%) and 4 (10.8%) were MDR and XDR phenotypes, respectively.

Of 8 isolates belonging to IC I, 6 isolates were MDR phenotypes with strong biofilm formation capacity and class I integron. Additionally, of 11 isolates belonging to group 10, 8 isolates had bla_OXA-23-like, 2 isolates had bla_OXA-23-like/bla_OXA-58-like, 1 isolate had bla_OXA-23-like/bla_OXA-24-like genes, and 3 isolates possessed bla_IMP/bla_VIM gene. In this group, all the isolates were resistant to imipenem, tobramycin, and levofloxacin.

Via the MLVA typing method, 70 A. baumannii isolates were grouped into 12 distinct MLVA types (MTs) with 5 clusters and 4 singleton genotypes. MLVA-AB_2396 showed a high level of diversity, whereas other MLVA allelic profiles indicated low diversity (Figure 1). The most common MTs were types III (20), IV (14), and XI (14). Among the isolates belonging to MT type III, 18 (90%) isolates had class I or II integron or both, and 16 (80%) isolates formed strong biofilm. Furthermore, all the 70 (100%) isolates were susceptible to colistin, and 8 (40%) isolates were from the ICU.

Figure 1.

MLVA-8 allelic profiles and characteristics of A. baumannii strains. Cut-off value of 90% similarity.

Discussion

Over the past years, the antibiotic resistance of A. baumannii has dramatically increased, and like many other geographical regions, our country (Iran) has been confronted with this major problem [28]. In this study, the results of Vitek-2 system regarding the resistance patterns of 70 A. baumannii clinical isolates against 17 antibiotics revealed that 87.1% and 12.8% of the isolates were MDR and XDR phenotypes, respectively.

So far, several studies have reported different frequencies of MDR and XDR A. baumannii isolates. In a study by Maspi et al. [29] in Tehran, the capital of Iran, the frequency rate of MDR and XDR A. baumannii clinical isolates were determined as 94.2% and 71.2%, respectively, which are higher than the rates reported in the current study. In another investigation in Bushehr City, in the south of Iran, Rahimi et al. [30] observed a frequency rate of 21% for MDR and 41% for XDR A. baumannii isolates. The discrepancies between these results likely come from the geographical differences, the diverse origin of bacterial isolates, and the lack of a standard MDR/XDR pattern definition template in various studies.

Our results suggested that the third- and the fourth-generation cephalosporins had no effect on A. baumannii isolates. The study of El-Mahdy et al. [31] conducted in Saudi Arabia reflected a 100% resistance rate against the aforementioned antibiotics, which was comparable with our finding. On the other hand, our study revealed a higher resistance rate of A. baumannii isolates against the third- and the fourth-generation cephalosporins compared to previous studies in Serbia [32], India [33], and Poland [34].

The results of antibiotic susceptibility testing disclosed that more than half of the A. baumannii isolates were resistant to the aminoglycoside family, among which amikacin with 84.2% resistance rate had the least effect. These observations were in line with that of Upadhyay et al. [35] who reported a high-level of aminoglycoside resistance (79.2%) in A. baumannii isolates recovered from patients admitted to the ICU of a tertiary referral hospital in Northeastern India. Besides, in some regions of Iran, high-level aminoglycoside-resistant A. baumannii clinical isolates (more than 70%) were reported by Mirnejad et al. [36] and Hatami Moghadam et al. [37] whose results were in agreement with the findings of the current study.

More than 70% of our A. baumannii isolates were resistant to carbapenems, which is in accordance with the findings obtained by Maspi et al. [29] in Tehran (Iran), Rynga et al. [38] in New Delhi (India), and Nowak et al. [39] who recorded high resistance rates for imipenem (96.9%) and meropenem (87.7%) in A. baumannii isolates collected from patients with ventilator-associated pneumonia in Greece, Italy, and Spain. However, reports from Poland and Japan displayed considerable lower resistance rates for carbapenems, which contradicts our finding that more than 50% of the isolates were susceptible to carbapenems [34,40].

The emergence of MDR and XDR carbapenem-resistant clones harboring oxacillinases and metallo-β-lactamases genes has raised considerable concerns for health systems worldwide. Here, the most prevalent oxacillinase was the bla_OXA-23-like (90%), a result similar to that of a former survey in which the bla_OXA-23-like was recognized as the most detected gene [32]. This observation was also in agreement with earlier reports from Iran and Mediterranean countries like Turkey that highlighted the bla_OXA-23-like gene as the most prevalent acquired oxacillinase in A. baumannii isolates [32,41]. The occurrence rate of 25.7% of bla_OXA-24-like gene in the present investigation was in contrast to the rate obtained in the study of Castilho et al. [42] who did not observe this gene in any CRAB isolates from Brazil. Another study from Tehran, Iran, showed a lower incidence rate of bla_OXA-24-like (1%) compared to the current research in which 11.4% of isolates harbored bla_OXA-58-like gene, while bla_OXA-143-like gene was not found in any isolates [43]. In agreement with our results, Sarikhani et al.’s [41] investigation from Qom, in north-central Iran, presented an occurrence rate of 55.6% and 14.4% for bla_OXA-58-like and bla_OXA-143-like genes, respectively. Until now, the emergence of new class D β-lactamases, bla_OXA-143-like and bla_OXA-235-like, in A. baumannii isolates was limited to Brazil, Mexico, and USA [44].

The metallo-β-lactamase genes were detected in 87.1% (61/70) of isolates. This finding in our study is supported by Soltani et al. [45] who reported the presence of 82.6% of these genes in A. baumannii isolates in the south of Iran. In the present research, 12 (17.1%), 18 (25.7%), and 22 (31.4%) isolates carried bla_SPM, bla_IMP, and bla_VIM genes, respectively, while no isolates were positive for the bla_NDM and bla_SIM determinants. Maspi et al. [29] pointed out that 2.3%, 15.1%, 2.3%, 4.7%, and 2.3% of isolates carried bla_VIM, bla_IMP, bla_SPM, bla_GIM and bla_SIM genes, respectively, which differs from our study. On the other hand, their results demonstrated that none of the tested strains harbored the bla_NDM gene. In the studies conducted by Amin et al. [46] and Gholami et al. [47] in Iran, the prevalence of bla_SPM gene was reported to be 5.19% and 3.63%, respectively. Pournajaf et al.’s [48] report from Iran and Gomma et al.’s [23] report from Egypt affirmed our finding that the bla_VIM is the most prevalent metallo-β-lactamase.

Our results are in accord with several previous studies showing that colistin and tigecycline are still among the most effective drugs in the treatment of MDR and XDR A. baumannii [32,33,43]. The resistance rate of 2.8% for colistin in this investigation was comparable with its global pooled prevalence of 0.0–3.7, while the resistance rate of 45.7% for tigecycline was higher than its global pooled prevalence of 2.3–25.8 [49]. Likewise, Salehi et al. [43] observed a higher resistance proportion (77%) for tigecycline than global pooled prevalence among clinical A. baumannii isolates.

The ability of A. baumannii to colonize and produce biofilm on biotic and abiotic surfaces is one of the main bases of chronic and nosocomial infections caused by this bacterium [50]. In our study, the results of MTP method revealed the capability of biofilm production by 92.8% of isolates, among which 78.5% were strong biofilm producers. Based on earlier evidence from Iran and Korea, the same as this study, more than 70% of isolates were able to produce biofilm [50,51]. Furthermore, the ability of biofilm production in A. baumannii isolates was not significantly correlated with resistance to antibiotics. Previously, Rahimi et al. [30] and Qi et al. [52] signified an inverse relationship between biofilm formation ability and antibiotic resistance or the acquisition of MDR/XDR phenotypes in A. baumannii isolates, while a study by Yang et al. [53] revealed that the MDR A. baumannii isolates provided a higher biofilm formation capacity.

PCR assay data indicated that 75.4% and 16.3% of isolates harbored class I and class II integrons, respectively, while class III integron was not identified in any isolates. Halaji et al. [54] reported a higher prevalence in Isfahan, Iran, for class II integron (78.2%) than class I (63.9%) in clinical A. baumannii isolates, which contradicts our results. Remarkably, a number of studies in this area expressed a higher prevalence for class I than class II integrons, but they could not detect class III integrons [54,55]. The lack of correlation between the presence of integrons and antibiotic resistance, except for the tetracycline, was another finding of our study. However, some previous investigations from different regions of the world emphasized that A. baumannii isolates harboring integrons had more potential to become MDR than integron-negative isolates [54–56].

The three-locus dual assay multiplex PCR and MLVA-8 were performed in this study to assess the clonality of A. baumannii isolates. The results of three-locus multiplex PCR suggested six different PCR-based or IC lineages (G1, G2, G4, G7, G10, and G15) among A. baumannii isolates, while Farshadzadeh et al. [57] reported eight different IC types. The G1 (IC II) with a frequency of 52.8% was the most predominant group in this study, which correlates strongly with the finding that the IC II is the most frequent lineage in the world [58,59]. The incidence of group G2 (CI I; 11.4%) was lower than the frequency (19%) recorded for this IC lineage [60]. In the current study, 35.8% (25/70) of isolates belonged to IC variants, which was lower than the rate reported by Bahador et al. [60] who indicated the occurrence rate of 43% for this lineage. Based on the results of MLVA-8, A. baumannii isolates were assigned to 12 different MTs, each had 1–20 members with the similarity cut-off of 90%. However, in another study in Iran, 32 MT variants were reported [60]. In our study, the MLVA-AB_2396 had the highest level of diversity, whereas MLVA-AB_0845 had the highest level of diversity in Iran and Spain [61,62].

In another study from France, Hauck et al. [56] reported the efficiency of an automatized MLVA-10 assay for the rapid and efficient genotyping of A. baumannii isolates. In our study, the MLVA-AB_2240 had an amplification failure for one isolate (Ab 66), while in other investigations in Iran [63] and France [56], Abaum3468, Abaum3002, Abaum3530, and Abaum0826 had an amplification failure, which is likely due to the partial deletion of the locus. Recently, Graña-Miraglia et al. [64] have shown that distantly related lineages can coexist in the same hospital in Honduras and Mexico. Our study supports the same pattern as diverse MDR A. baumannii populations are co-circulating in the Southwest of Iran, including the Imam Khomeini Hospital in Ahvaz; importantly, this pattern makes it difficult to properly implement infection control policies. In this research, due to financial limitations, the sequencing of the MBL genes was not performed.

Conclusion

This study documented a high distribution rate of MDR A. baumannii isolates, harboring different metallo-β-lactamase and oxacillinase genes, in one of the main regions in the southwest of Iran. Significant resistance to most antibiotics, except for colistin and tigecycline, in A. baumannii isolates seems to pose a serious challenge to healthcare systems in the future. The diverse A. baumannii lineages in this study highlights the need for designing appropriate antibiotic surveillance programs in each region based on the predominant circulating clone in order to choose the best therapy and manage infection control policies.

Disclosure statement

The authors declare that they have no conflict of interest in this study.