Iranian Journal of Medical Microbiology

Isolation and Identification of Isolates

A total of 50 isolates of A. baumannii were randomly selected from the microorganisms collection of the Iran University of Medical Sciences. These bacteria were selected from the clinical samples of the respiratory system of patients who referred to Imam Khomeini Hospital, Tehran, Iran during 2018-2019 [9]. Biochemical tests, including oxidase, catalase, anaero-bic oxidation-fermentation (OF), aerobic OF, triple sugar iron (TSI), indole, motility, culture on Simmons Citrate agar, Voges-Proskauer, methyl red, culture on MacConkey agar, and lactose test (Merck, Germany) were performed to ensure correct colony selection. Finally, 50 A. baumannii isolates were identified and confirmed [10].

Primers Designing

The target sites for A. baumannii primers were chosen as nine specific genes, namely bap, ompA, basD, espA, bfmR, lpD, csuA, pbpG, and surA. Following the determination of target sites for primer designing, databases of each bacterium were searched on NCBI. The Oligo 2, Oligoanalyzer, and Gene Runner softwares were applied to assess the annealing and Tm temperatures of primers (Table 1). Next, blasting with human, fungal, viral, and microorganism samples was performed with NCBI databases for the designed primers.
 
Table 1. Sequences of primers used for identifying specific genes in A. baumannii isolates  

DNA Extraction from A. baumannii Isolates

First, colonies of all A. baumannii isolates were separately cultured in microtubes containing 2 mL of Tryptic Soy Broth (TSB) medium and were incubated at 37°C for 24 h, which followed by the extraction of genomic DNA from colonies by the boiling method.
First, bacterial suspension was centrifuged at 10000 rpm for 10 min after 24 h incubation. Afterwards, 1 mL of the supernatant was discarded and 1 mL distilled water was added to the bacterial precipitate. This procedure was performed twice. Next, 200 µL of TE sterile buffer (pH: 8) was added to the bacterial precipitate. Microtubes containing precipitate and TE buffer were boiled at 100°C for 10 min and were immediately transferred to the temperature of -20°C for 5 min. The process of boiling and cooling was repeated three times. Afterwards, the microtubes that contained bacterial precipitation were centrifuged at 14000 rpm. Finally, the supernatant containing genomic DNA was collected in a sterile microtube for polymerase chain reaction (PCR). In order to determine the purity of extracted DNA, electroph-oresis and biophotometer were carried out.

Multiplex PCR

PCR was performed using Taq DNA Polymerase Master Mix (Amplicon, Denmark) which consists of MgCl₂, dNTPs, Taq polymerase, and buffer. The final volume of each reaction for completing multiplex PCR was 25 µL containing 12 µL Master Mix for all reactions. Moreover, 0.3 µL at the concentration of 10 pM was poured in a microtube from each forward and reverse primer of ompA gene with 1 µL of both primers for bap gene. In addition, 0.5 µL of both reverse and forward primers of the three genes csuA, plD, and basD were poured into another tube. 
The amounts of 1, 0.5, and 0.5 µL of the reverse and forward primers of pbpG, bfmR, surA genes were added to the third microtube, respectively. A volume of 0.5 µL of each primer of the espA gene was poured into another tube. The espA gene was evaluated separately and not as multiplex. The final concentration for each primer was 100 µmol. After that, 1 µL DNA at the concentration of 200 ng/L and 1 µL double-distilled water free of any nuclease was added to each reaction mixture to reach the intended final volume.
Multiplex PCR was carried out as follow: 1) primary denaturation for 5 min at 95°C, 2) 34 denaturation cycles of 40 sec at 95°C, 3) annealing for 45 sec (annealing temperature was determined as 59°C for all nine genes, 4) polymerization for 45 sec at 72°C, and 5) final polymerization for 5 min at 72°C. Afterwards, PCR products were electrophoresed on 1% agarose gel containing safe stains and were visualized by gel documentation. The PCR products were finally sent to Pishgam Co. (Iran) for sequencing.

Statistical Analysis

Statistical analysis was performed using the SPSS software version 22 (IBM, Chicago, Ill., USA). The relationship between antibiotic resistance and the frequency of ompA, bap, bfmR, espA, basD, surA, pbpG, csuA, and plD was assessed by the t-test.

During the last decade, A. baumannii has been identified as the most important nosocomial pathogen throughout the world. It is partly due to the living ability of this microorganism in the hospital environment and obtaining resistance mechanism leading to acute infections, especially in severely ill patients. Although extensive information exists concerning the mechanisms of antibiotic resistance in this microorganism, it cannot be stopped yet [11].
Although the severity of A. baumannii infection is directly related to the pathogen factors, the frequency of these factors in clinical samples of A. baumannii is less reported [12]. Therefore, the infection caused by this agent and the immune response of the host to these infections is still ambiguous. In the present study, the frequency of nine genes of virulence factors were evaluated in the clinical samples of A. baumannii isolated from the respiratory system of patients. Biofilm is considered as an important factor in clinical specimens of A. baumannii. The extension and thickness of biofilm structures and intracellular attachments are tightly related to bap family proteins, which are coded by the bap gene [13, 14].
Our results concerning the frequency of pathogen genes were highly similar to the findings of Chaoliu et al. in China. In the mentioned investigation, the frequency of the bap gene in the clinical samples of A. baumannii was reported as 87.5%. Furthermore, Fallah et al. in 2019 found the frequency of 92% in 100 clinical isolates in Iran [15, 16].
The ompA is a protein of the outer membrane that induces biofilm formation on the epithelial cells of humans. This protein is coded by ompA [5]. In Iran, Bardbari et al. (2017) and Zeighami et al. (2019) reported the frequency percentage of this gene as 100% in clinical isolates of A. baumannii isolated from respiratory samples and 81% in the isolates related to other clinical specimens, respectively [17, 18]. Their results were highly similar to the findings of the current investigation (96%). Although the ompA gene has a high frequency, previous studies have shown that biofilm formation is more associated with the bap gene, compared to the ompA gene [19].
The csuA gene belongs to the csu gene operon, which consists of six genes (csuA/BABCDS) and codes the chaperone-usher secretory system as a very important part of pilli generation and biofilm structure formation on non-living surfaces [20]. The bfmR gene as the regulatory part of the two-component regulating system had a frequency of 100%. The pres-ence of this gene in A. baumannii isolates is essential for pilli generation and biofilm formation on plastic surfaces [6]. Thummeepak et al. in a study in Thailand (2016) on A. baumanni clinical samples reported a frequency of 84% for the latter gene [5]. These findings revealed that most of the clinical isolates of A. baumannii had the potential for biofilm forming. The frequency of the espA gene in the present study (10%) was lower than the other genes that contribute to biofilm formation, including csu, bfmR, espA, ompA, and bap.
Thummeepak et al. and Kim et al. (2019) observed the frequencies of 30% and 50% for the mentioned gene in 255 clinical samples and 181 specimens isolated from blood culture in South Korea, respectively [5, 21]. Two other genes investigated in the current study were plD and surA, both of which are serum resistance factors [7, 22]. Chaoliu et al. reported the frequency of 95% for surA and 92% for plD, which is consistent with our results [15].
The basD was another gene with a frequency of 96% in the current study. Chaloliu et al. and Kim et al. revealed it to be 92% and 98%, respectively [15, 21]. Finally, the pbpG gene that codes PBPs had a frequency of 100% in A. baumannii isolates. Although PBPs are the receptors of beta-lactam antibiotics, are known as one of the pathogen factors in A. baumannii isolates and account for bacterial survival in human serum and the soft tissues of animal models [20].
The findings of some investigations had a very low similarity with our results. The latter discrepancy could be attributed to the type of studied samples, study time, and the type of used antibiotic disk. On the other hand, these bacteria benefit from diverse mechanisms for pathogenicity, scape host immune system, and antibiotic resistance. Numerous studies have been performed in Iran on the epidemiologic characteristics and drug resistance pattern of A. baumannii isolates. However, it is necessary to consider the role of this bacterium as a potentially dangerous factor in nosocomial infections.

The genes involved in the pathogenicity of A. baumannii have a high frequency and the nosocomial prevalence of this bacterium has elevated. Therefore, large-scale studies and protection programs, such as infection control in the ICU seem to be necessary. Limiting the consumption of antibiotics, development and changing of hygiene plans for contaminated facilities, and classifying patients in whom this bacterium has colonized are among the beneficial approaches for controlling the spread of A. baumannii.

The present paper is related to the Master thesis of the first author.

The current study has financially been supported by the Research Deputy of Iran University of Medical Sciences with the grant code of 98-4-4-16126. The authors would like to thank the latter deputy.
 
 

Conflicts of Interest

The authors declared no conflict of interest.