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Prevalence Determination of Virulence Related and Biofilm Formation Genes in Acinetobacter baumannii Isolates from Clinical Respiratory Samples in Imam Khomeini Hospital, Tehran, Iran in 2018

Background and Aim: Acinetobacter baumannii is considered to be a re-emerging causative agent of nosocomial infections. There is a significant relation between pathogenicity of this bacterium and the numerous virulence factors. The purpose of this study was to investigate nine virulence factor genes in A. baumannii isolates derived from hospitalized patients. Materials and Methods: A total of 50 A. baumannii isolates were recovered from patients with pneumonia in Imam Khomeini Hospital, Tehran, Iran. Following biochemical and microbiological identification of the bacteria, Multiplex PCR was performed for basD, plD, csuA genes, surA, pbpG, bfmR genes, and bap, ompA genes using specific sets of primers which were specifically designed for this study. The espA was identified separately by a Uniplex PCR assay. All amplified DNA fragments were sequenced for the products’ confirmation. Results: Among the 50 clinical isolates of A. baumannii studied, bfmR and pbpG genes were reported in all samples (100%), bap, plD, surA, and csuA genes were collected from 49 samples (98%), 48 (96%) of these isolates had ompA and basD genes, and espA gene was observed in only five isolates (10%). Conclusion: According to this study results, virulence factors genes in clinical A. baumannii have a prevalence rate more than 90%. Additionally, the high incidence rate of those genes related to biofilm formation indicates that most clinical strains have the ability to form biofilm structures.


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, electrophoresis 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.

Identification of A. baumannii
Microbiologic and biochemical tests were performed for the definite determination of 50 A. baumannii isolates collected from the respiratory system of patients. In the laboratory, all 50 isolates were cultured on MacConkey agar and blood agar and were incubated at 37°C for 24 h. After that, the presence of Gram-negative coccobacilli A. baumannii was confirmed by a microscope. Next, biochemical tests, such as IMViC, urease, TSI, OF, MR, VP, SIM, catalase, oxidase, and growth at 37°C and 42°C were conducted to diagnose diverse species of Acinetobacter.
Isolates that were lactose negative, non-motile, oxidase negative, catalase positive, indole negative, pigmentation negative, urease positive, citrate positive, H2S negative, and MR negative were confirmed. The results of microbiologic and biochemical tests demonstrated that all 50 samples were A. baumanni and stored at -70°C in Peptone Water medium containing 30% glycerol.

Results of PCR for Specific Genes
All samples identified as A. baumanni by biochemical tests were tested by multiplex PCR for ompA, bap, bfmR, plD, basD, espA, csuA, pbpG, and surA genes. The results of multiplex PCR revealed that the genes bfmR, bap, ompA, and csuA involved in biofilm formation had the frequencies 100%, 98%, 96%, and 98%, respectively. Gene espA was found to have the lowest frequency of 10% among the isolates. The lengths of products are demonstrated in Table 1.
According to the findings of previous studies, the bap gene is the most important gene for biofilm formation [5]. The frequency of this gene was very high in the current study (98%) and only one isolate lacked this gene. The frequency of other investigated genes in the present study was 98%, 96%, 98%, and 100% for plD, basD, surA, and pbpG, respectively (P<0.05) (Figures 1-5). For all negative specimens, PCR was repeated twice as a single gene. The frequency of genes basD, bfmR, bap, ompA, plD, csuA, pbpG, and surA was 82% ( Table 2). The PCR products were finally sent to Pishgam Co. for sequencing. The findings of sequencing showed a similarity of over 80% in isolates (P<0.05) ( Figure 6).

Discussion
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. 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 presence of this gene in A. baumannii isolates is essential for pilli generation and biofilm formation on plastic surfaces 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.

Conclusion
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.

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

Financial Support
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.