Antibiotic Resistance Patterns and Distribution of Ampc β-Lactamases Genes Among Acinetobacter Baumannii Clinical Isolates from Hospitals of Tehran, Iran

Acinetobacter is a Gram-negative coccobacilli bacterium that can produce severe and different infections. Among the species of these bacteria, Acinetobacter baumannii is the most common cause of nosocomial infections. Due to the high prevalence of antibiotic resistance in this bacterium and the signicant increase in antibiotic resistance, this study was conducted to investigate the antibiotic susceptibility of A. baumannii isolates from Iran. A total of 60 A. baumannii bacteria were isolated from the different clinical samples in hospitals of Tehran, Iran. The isolates susceptibility to 13 commonly used antibiotics was examined according to the Clinical & Laboratory Standards Institute (CLSI) guidelines. Using PCR, three important AmpC β-lactamases relayed genes (DHA, CIT and MOX) were detected.

To treatment of A. baumannii infections, various antibiotics, including β-lactams, uoroquinolones, and aminoglycosides, are used. Today, many studies have shown that antibiotic resistance in the A. baumannii isolates is extremely increasing [3,4,6,7]. Inappropriate and indiscriminate use of available antibiotics, high ability of natural genetic transformation in A. baumannii bacteria and its potential for widespread dissemination are most important factors contributed to the resistance of this pathogen to the various antimicrobial agents [8,9].
Currently, β-lactam antibiotics play critical roles in the treatment of A. baumannii infections. But from 1991 until now there are many studies that reported the resistance of A. baumannii to this class of antibiotics [5]. Molecularly, resistance to β-lactams in A. baumannii may be due to the activity e ux pumps, changes in penicillin-binding proteins (PBPs), and the production of antibiotic-degrading enzymes such as production of β-lactamases [10].
According to these facts the present study was aimed to evaluate the phenotypic and genotypic antibiotic resistance pattern of A. baumannii clinical isolates and comparison of results to the previous described ndings. In addition, we investigated the presence of AmpC β-lactamases enzyme production and also AmpC β-lactamases related genes.

Demographic data
A total of 60 A. baumannii isolates were isolated from 240 clinical isolates were obtained based on applied differential and biochemical tests. These isolates were obtained from 27 (45%) women and 33 (55%) men. Among them, 6 (10%) cases hospitalized due to abdominal surgery, 7 (11.66%) cases due to heart surgery, 2 (3.33) cases due to brain surgery and 11(18.33%) cases due to respiratory problems.
These data are presented in detail in Table 1.

Antibiotic resistance patterns
Our results revealed that out of 15 studied antibiotics, the resistance rate above 90% was reported in 12 cases. Based on our results the tested isolates are highly resistant to the ampicillin (99%), cefotaxime (98%), chloramphenicol (98%), ceftriaxone (98%), ceftazidime (98%), meropenem (98%) and ticarcillin (98%). On the other hand, tobramycin and colistin with 78.3% and 35% resistance rates were the most effective antibiotics, respectively. The results of the present study showed high resistance rate of clinical isolates to tested antibiotics. The total resistance pattern was presented in Table 2. In this study, out of 60 isolates, 59 isolates (98.34%) were resistant to more than 8 antibiotics and only 1 sample (1.66%) which isolated from urine culture was sensitivity to all studied antibiotics.

Distribution of Amp β-lactamases genes
The prevalence of DHI, MOX and CIT were 1 (1.66%), 27 (45%) and 7 (11.66%), respectively. Twenty-four isolates (40%) were negative for all three genes. There was a signi cant relationship between the presence of MOX gene and antibiotic resistance (p < 0.05). In other word, MOX-containing isolates were resistance to more tested antibiotics.

Discussion
In the past 2 decades, A. baumannii has become an important pathogen related to nosocomial infections and has been shown to increase morbidity and mortality. Control of infections caused by this bacteria is always di cult, due to its acquisition of multidrug-resistant phenotypes, such as resistance to uoroquinolones, aminoglycosides, cephalosporins, and carbapenems [1].
In the present study, the antibiotic resistance pro le as well as the phenotypic and genotypic AmpC βlactamases resistance was investigated. Our results revealed that out of 15 studied antibiotics, 12 antibiotics had a resistance above 90%. The most resistance rate was related to ampicillin (99%), cefotaxime (98%), chloramphenicol (98%), ceftriaxone (98%), ceftazidime (98%), meropenem (98%) and ticarcillin (98%). These results can be more important in related to the antibiotics which are used to treatment of A. baumannii infections; such as third-and fourth-generation cephalosporins and carbapenems [2].
The results of this study are in line with the ndings of previous studies which reported that most strains of A. baumannii isolated in Iran were resistant to rst-line drugs including aminoglycosides, uoroquinolones and carbapenems [14][15][16]. In a study conducted by Karimi et al (2020) on 60 A. baumannii isolates obtained from Hazrat-e-Rasoul Hospital in Tehran, the highest resistance of isolates was related to ceftazidime (93.3%) and amikacin (91.6%), respectively. In their study, colistin with 3.3% resistance was introduced as the most effective antibiotic [17]. In terms of antibiotic resistance pro le, the results of this study were in line with our present study.
Due to rising antimicrobial resistance, carbapenems are the cornerstone of therapy for the treatment of A. baumannii serious infections [18]. However, the results of the present study showed a high prevalence of meropenem resistance (96.7%), which has increased signi cantly compared to previous studies in different cities of Iran and also other countries [19,20]. In similar studies in Tehran, 50.9%, 52.5%, 62% and 67.5% of the isolates were resistant to imipenem and 51.8%, 52.5%, 62% and 84.5% to meropenem in 2008, 2009, 2011 and 2013, respectively [16, [21][22][23].
The analysis of these data and comparing them with the results of the present study results reveals that antibiotic resistance of A. baumannii isolates to carbapenem antibiotics were increasing in Iran over the time.
In our study low susceptibility rates to most of available antimicrobial agents for the treatment of A. baumannii isolates was seen, except for colistin. Although several number of previous studies conducted in Iran have shown that all of the A. baumannii isolates were sensitive to colistin [15,21,24], but in the present study, 35% of the isolates were resistant to colistin. Given that drug resistance rate of A. baumannii to colistin was not high, this antibiotic can be administered as appropriate therapeutic drug against A. baumannii infections.
In contrast to the present study which found PDR and XDR as well as MDR A. baumannii isolates, Hojabri (2014) only found MDR strains among the isolates [26]. In Bahador et al (2015) study, the frequency of MDR, XDR and PDR isolates were 69, 24, and 0%, respectively [27]. In similar study performed by Sobouti et al (2020) in Iran, from 62 A. baumannii, 36 (58%) strains were categorized as MDR, 17 (27.5%) as XDR, and 9 (14.5%) as PDR [25]. Compared to the results of this study, the percentage of resistant strains in the present study was higher. Comparison of these results and also similar studies shows that the emergence of resistant strains in A. baumannii isolates is increasing in Iran and the strict control measures should be considered in this subject.
In next part of study the prevalence of some AmpC β-lactamases related genes (FOX, DHA and MOX) in A. baumannii isolates were detected. Based on these results, it was found that out of 60 isolates, 1 (1.66%), 27 (45%) and 7 (11.66%) isolates were positive for DHI, MOX and CIT, respectively. Consistent with the present study, in Fekri et al (2017) study AmpC β-lactamases related genes detected in 60 strains A. baumannii isolates using Multiplex PCR, of which 39 (65%) had CIT, 36 (60%) had DHA and 12 (20%) had MOX gene [28]. The products of these genes were consistently associated with resistance or at least reduced susceptibility to a wide variety of β-lactam antibiotics. For this reason the presence of these genes in the individual isolate is very important [29]. Our results also shown that, there is some cephalosporins-resistant isolates which are negative for the some investigated genes. Resistance to these antibiotics in those isolates may be due to other mechanisms including decreased permeability, alteration of PBPs, presence of other AmpC related genes and overexpression of e ux pumps [30].

Conclusion
This study has shown that resistance to the majority of antibiotics in the A. baumannii strains is high with 59 (88.33%), 44 (73.33%) and 13 (21.66%) isolates were MDR, XDR and PDR, respectively. High rate of resistance to cephalosporins has been seen among our isolates and it seems that, colistin can be effective drug for treatment of A. baumannii infections. Progressive increase in resistance to the majority of antibiotics and multiple resistances in the present study may be related to increased usage of these antibiotics for treatment of A. baumannii infections. Management of treatment, such as performing of antibiogram test before antibiotic therapy for select the appropriate antibiotic and also completes the course of treatment period is necessary to prevent the further spread of resistant isolates.

Bacterial isolation
A total of 240 clinical samples (including blood, urine, sputum, respiratory secretions, urine, wounds and skin) were collected from patients admitted to different wards of hospitals in Tehran between 2018 to 2020. Samples were collected from patients who had been hospitalized for at least three days and who had received the infection from the hospitals. A. baumannii were identi ed using Gram staining, culture characteristics on the differential culture media (blood agar, MacConkey agar, Triple sugar iron agar (TSI) and oxidative-fermentative (OF) agar and biochemical tests (catalase and oxidase) [31]. The isolated bacteria were stored in Tryptic Soy Broth (TSB; Merck, Germany) containing 30% glycerol at -70°C until further analysis.

Antibiotic Susceptibility Testing
The antibiotic susceptibilities testing for A. baumannii isolates were done by Kirby Bauer's disk diffusion method on Muller-Hinton agar (Merk, Germany) according to the CLSI [32]. The applied antibiotic disks (MAST, UK) were ampicillin (25 µg Determination of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates According to the CLSI, MDR was de ned as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was de ned as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories for example bacterial isolates remain susceptible to only one or two categories and PDR was de ned as non-susceptibility to all agents in all antimicrobial [33].

Dna Extraction And Multiplex Pcr
DNA extraction was carried out from 10 ml overnight cultures of each isolate in tryptic soy broth (TSB) by Cinnagen extraction kit (Cinnagen, Iran) according to the manufactory instructions. Extracted DNA samples from all of the isolates were examined by the PCR assay for three target genes, MOX, CIT and DHA.
The oligonucleotide primers were previously described by Hanson et al [34] and their sequences are given in Table 3. For each gene, the nal volume of the reaction mixture was 25 µl contained 3 µl of template DNA, 2.5 µl of ×10 PCR buffer, 0.5 µl of 10 mM dNTPs, 0.75 µl of 50 mM MgCl2, 0.25 µl of 5 U/µl of Taq DNA polymerase, and 25 pmol of each used primer.
The PCR ampli cation was performed under the following thermal conditions: initial denaturation at 94°C for 1 min followed by denaturation at 94°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1.5 min (30 cycles), and a nal extension at 72°C for 10 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide.