Molecular study of metallo-β-lactamases and integrons in Acinetobacter baumannii isolates from burn patients

Background Productions of metallo-β-lactamases enzymes are the most common mechanism of antibiotic resistance to all beta-lactam classes (except monobactams) in Acinetobacter baumannii. MBLs are usually associated with gene cassettes of integrons and spread easily among bacteria. The current study was performed to detect the genes encoding MBLs and integron structures in A. baumannii isolates from burn patients. Methods This study was performed on 106 non-duplicate A. baumannii isolates from burn patients referred to Shahid Motahari Hospital in Tehran. Antibiotic susceptibility of A. baumannii isolates was performed using disk diffusion and broth microdilution method in accordance with the CLSI guidelines. The presence of class 1 integron and associated gene cassettes as well as MBLs-encoding genes including blaVIM, and blaIMP were investigated using PCR and sequencing techniques. Results In this cross-sectional study all (100%) of the A. baumannii isolates examined were multidrug resistant. All isolates were sensitive to colistin and simultaneously all were resistant to imipenem. PCR assays showed the presence of blaVIM and blaIMP genes in 102 (96.2%) and 62 (58.5%) isolates of A. baumannii respectively. In addition, 62 (58.5%) of the A. baumannii isolates carried integron class 1, of which 49 (79.0%) were identified with at least one gene cassette. Three types of integron class 1 gene cassettes were identified including: arr2, cmlA5, qacE1 (2300 bp); arr-2, ereC, aadA1, cmlA7, qacE1 (4800 bp); and aac(3)-Ic, cmlA5 (2250 bp). Conclusion A high prevalence of MBLs genes, especially blaVIM, was identified in the studied MDR A. baumannii isolates. In addition, most of the strains carried class 1 integrons. Furthermore, the gene cassettes arrays of integrons including cmlA5 and cmlA7 were detected, for the first time, in A. baumannii strains in Iran.


Background
Burn infections are a noticeable health problem, especially in developing countries [1]. It has been documented that about 75% of death in patients with burn injuries are due to infections [2] Acinetobacter baumannii is one of the most common causes of nosocomial infections with high mortality and morbidity among hospitalized patients, especially in burn and intensive care units [3,4]. Nowadays, the emergence and spread of antibiotic resistance in A. baumannii is a major global challenge [4]. Carbapenems are considered drugs of choice for the treatment of severe infections caused by MDR-A. baumannii [5]. Unfortunately, carbapenem resistance is increasing among A. baumannii isolates, which is alarming [5].
Various mechanisms are involved in the development of carbapenem resistance in A. baumannii including β-lactamases acquisition, outer membrane proteins and PBPs alteration, overexpression of efflux pumps and gene mutation of CarO [6].
One of the most important mechanisms of antibiotic resistance in A. baumannii is the production of β-lactamase enzymes, the genes of which are usually carried on mobile genetic elements, including integrons [7]. Beta-lactamases are grouped into four classes based on the amino acid sequence, including: A, B, C, and D [8]. Resistance to carbapenems is usually dependent on β-lactamases of class B (MBLs) and D (OXA-type carbapenemases) [9]. OXA β-lactamases or OXA-type carbapenemases, include distinct subgroups from which OXA-23-like, OXA-24-like, OXA-40-like, OXA-51-like, OXA-58-like and OXA-143-like have been found in A. baumannii strains [9,10]. MBLs families are more important than other β-lactamases, due to their ability to hydrolyze a wide range of β-lactam antibiotics, especially carbapenems [11,12]. Several MBLs including VIM and IMP have been identified among A. baumannii strains [13,14]. Different IMP-type enzymes have been described in the globe among Gram-negative bacilli especially Enterobacterales and non-fermenter organisms including Acinetobacter spp. [15]. Previous studies have shown that the prevalence of MBL-producing strains of A. baumannii in the world is increasing, although there are different reports in various geographical areas [13].
Acinetobacter has a high potential for the acquisition of resistance genes through mobile genetic elements, including integrons [15]. MBLs are generally encoded on the gene cassettes of class 1 integrons and spread readily among A. baumannii strains [16]. The presence of integrons and association with genes encoding MBLs are frequently reported in A. baumannii isolates [17].
The current study was performed to detect the genes encoding MBLs and class 1 integron structures in A. baumannii isolates from burn patients.

Ethical consideration
Informed consent was obtained from all subjects, and all methods were carried out in accordance with the relevant guidelines and regulations of Ethics Clearance Committee of the Alborz University of Medical Sciences.

Bacterial isolates and identification
The current study was conducted between December 2019 and November 2020. A total of 106 non-duplicate A. baumannii isolates were collected from hospitalized burn patients at Shahid Motahari Hospital in Tehran, Iran. The collected clinical isolates were transferred to the laboratory of the Department of Microbiology, School of Medicine, Alborz University of Medical Sciences. Standard biochemical tests were used to identify the Gram-negative bacilli isolates as A. baumannii strains These tests included catalase, oxidase, O/F test, motility, citrate utilization test and growth on TSI agar (Merck, Germany) [18]. The diagnosed A. baumannii strains were cultured in TSB (Merck, Germany) supplemented with 20% glycerol and stored at − 20 °C for further studies. The phenotypically isolated A. baumannii strains were confirmed by PCR and sequencing of the rpoB gene [19]. The A. baumannii ATCC 19606 was used as a control strain.

Determination of integrons and associated gene cassettes
Extraction of genomic DNA of A. baumannii strains was performed using boiling method [22]. The presence of integrons class 1 and related gene cassettes were determined by PCR using related primers [23]. The gene cassettes of integrons were amplified using primers and PCR conditions as described previously [23,24]. Amplified gene cassettes were sent for sequencing (Macrogen Research, Seoul, Korea). The sequences obtained were compared with those deposited in the NCBI database with using BLAST program (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) to determine the cassettes arrays of integrons.

Molecular detection of metallo-β-lactamases-encoding genes
The amplification of the metallo-β-lactamases-encoding genes including bla VIM , and bla IMP which is associated with carbapenem resistance was performed by PCR assays in all isolates. The relevant primers used for PCR testing are listed in Table 1. The PCR conditions were performed according to the protocol provided [22].

Statistical analysis
All statistical analysis was performed using SPSS software version 21 (SPSS, Inc.). The differences between variables were evaluated by the chi-square (χ 2 ) test and P-values (P < 0.05) were interpreted statistically significant.

Results
In this cross-sectional study, a total of 106 non-duplicate A. baumannii isolates were collected from burn wounds of hospitalized patients. Seventy-six of the A. baumannii strains were isolated from male (71.7%) and 30 females (28.3%) patients. According to the results of antimicrobial susceptibility testing, all (100%) of our A. baumannii isolates were identified as MDR and 101 (95.3%) were XDR, however, PDR phenotype were not detected in any of the isolates. The antibiotic susceptibility profiles of the A. baumannii isolates by disk diffusion method are shown in Table 2. Determination of MICs of imipenem and colistin showed that all isolates were sensitive to colistin (MIC of ≤ 2 μg/ml) and at the same time all were resistant to imipenem (MIC of ≥ 8 μg/ml).
Interpretation of the results of statistical analysis showed there was no significant association between the presence of class 1 integrons and age groups (P = 0.55), and burn size (P = 0.52). However the presence of class 1 integrons were significantly higher in female than male patients (P = 0.017).

Discussion
Acinetobacter baumannii is one of the most important pathogens leading to infections in burn patients. Control of A. baumannii infections in these patients is a major challenge due to the proliferation of MDR strains [25]. In the current study, all isolates were identified as MDR strains. Also in accordance with other studies, we found that all our A. baumannii strains were carbapenem resistant [26]. Similarly to our study, 94.5% of A. baumannii isolated from burn patients were resistant to carbapenems in the study by Pournajaf et al., [5], suggesting that the mentioned group of antibiotics is no longer suitable for the treatment of infections caused by this bacterium. Determination of resistance by MIC showed that all strains were sensitive to colistin. These data are in agreement with those of Tarashi et al., [27] and previous studies [17], and show that colistin is still an effective antibiotic against MDR A. baumannii. In our study, bla VIM was identified as the most common gene encoding MBLs in the vast majority of isolates, followed by bla IMP . According to the literature, a wide distribution of VIM type metallo-β-lactamase has been reported at Middle East CRAB [22]. An association between class 1 integrons and MBLs genes, particularly bla IMP and bla VIM , has been reported in other studies [17]. However, our findings showed that the bla IMP and bla VIM genes were not located on the class 1 integrons and no association was found between MBL genes and the presence of class 1 integrons among studied isolates. The reason could be that these genes may have been located on the other region of bacterial DNA.
The analysis of integrons content revealed that 79.0% of studied integron class 1 positive A. baumannii strains carried at least one gene cassette, while 21.0% had no cassettes and carried empty integrons. Considering that strains with empty integrons have the potential to capture cassettes carrying resistance genes, this result could be remarkable. The most common integron cassettes identified among integron-positive A. baumannii strains were arr2, cmlA5, qacE1, which encode rifampin ADP -ribosyltransferase, chloramphenicol transporter and quaternary ammonium resistance protein leading to resistance to rifampicin, chloramphenicol and quaternary ammonium compounds, respectively. Rifampin resistance due to the arr-2 gene carried by class 1 integrons has been documented in A. baumannii strains [28]. Also other variants of cmlA gene have been reported in other studies and it seems that this is the first report of detecting cmlA5 and cmlA7 variants in A. baumannii strains in Iran [24]. Other cassettes, including aadA1 and aac(3)-Ic encoding aminoglycoside adenylase and aminoglycoside acetyltransferase respectively were found to be less common in the present study. In researches conducted in other parts of the world, including Taiwan, mainland China, and France, different variants of the aadA gene such as aadA1, aadA2 and aadA6 have been identified in multidrug-resistant A. baumannii strains [23,24,29]. The identification of aadA1 and aac (3)-Ic genes in cassettes of class 1 integrons could explain the high resistance to aminoglycosides in the present study. Despite the high resistance to antibiotics such as ceftazidime, ciprofloxacin, and trimethoprim/sulfamethaxazole, no gene cassette encoding resistance to these antibiotics was found in the present study. Therefore, the development of resistance to mentioned antibiotics may depend on the presence of genetic elements other than integrons.

Limitations
This project is a cross-sectional study. However, to demonstrate the clinical relevance and dynamic community structure of clinical isolates, we need to continuously monitor the outbreaks using PFGE, MLST, or WGS tools. More information about resistant bacteria helps us to respond to how bacteria spread throughout clinical settings. Part of the limitations of the current study is also due to limited resources. However, other genes responsible for carbapenem resistance and MBL formation could be identified by WGS. In addition to class 1, detection of gene cassette arrays associated with other integron classes could level up the study. Seemingly, the data obtained in the present study may provide a basis for future studies and assess the trend of infection generated by A. baumannii in burn patients in our region.

Conclusion
A high prevalence of MBLs genes especially bla VIM was identified in studied MDR A. baumannii isolates. In addition, most of the VIM type MBL-positive strains carried class 1 integrons. Furthermore, the gene cassettes arrays of integrons including cmlA5 and cmlA7 were detected, for the first time, in A. baumannii strains in Iran.