Evaluating the Activity of Ultrasound on Biofilm Formation by Acinetobacter baumannii isolated from clinical Specimens

: Acinetobacter baumannii received attention for its multi-drug resistant associated with many severe infections and outbreaks in clinical environment. The aims of the study are to investigate the antibiotic susceptibility profile of clinically isolated A. baumannii , biofilm production, and the efficiency of Low Frequency Ultrasound (LFU) and honey to attenuate biofilm production. A total of 100 samples were taken from different sources from Baghdad hospitals. The susceptibility patterns revealed the percentage of pan drug resistant (PDR) isolates were 1.5 %, 72.7 % were extended drug resistant (XDR), 16.7 % were multidrug resistant (MDR), and 9.1 % were non MDR and sensitive to most antibiotics used. The ability to form biofilm was detected by crystal violet staining, and the results showed that 20% were strong biofilm, and 31.8% were moderate biofilm. The biofilm formation percentage was decreased using Low frequency ultrasound LFU and honey. Moreover, PCR results revealed that not all of them harbouring biofilm-related genes or integrons ( bap , csu E, IntI-1, IntI -2), although, they are strong biofilm producers. These results conclude that low frequency ultrasound and chemical components of honey might be a good choice to restrain A. baumannii biofilm formation, and negative correlation between antibiotic resistance and biofilm ability


Introduction:
A. baumannii is a Gram-negative pathogen, aerobic non-motile and non-fermentative coccobacillus, it is strictly aerobic and is able to persistent in heterogeneity of the surrounding environment. A. baumannii also known as "Iraqibacter", causes a major problematic infection among soldiers through Iraq and Afghanistan war 1

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A. baumannii is a major nosocomial microorganism that causes a wide range of diseases in hospital setting, such as meningitis, wound infection, endocarditis, and bacteremia in mostly ICU patients. It also causes a ventilator-associated pneumonia (VAP), catheter-associated bloodstream, and urinary tract infections 2 .
Nowadays, A. baumannii is largely coupled with a significant infection among patients 3 , as it exhibits a tremendous resistance against practically all known antibiotics as well as to a host immune response, which makes it a crucial concern in treatment and public health 4 The efficiency of A. baumannii to form biofilm and it's persistence in dry and desiccated surrounding conditions generated problems and outbreaks in health-care treatment options, particularly for ill patients in the intensive care units (ICUs) 5 . Biofilm is the aggregation of bacterial cells embedded in an extracellular polymeric substances (EPS), called matrix, which enable bacterial cells to attach to various abiotic and biotic surfaces and protect their growth from the immune system and antibiotics 6 . Because biofilm formation is a multistep processes, several factors contribute to the formation and establishment of biofilm, bap gene is the biofilmassociated protein which displays an indispensable role in the formation of biofilm, intercellular adhesion and aggregation of bacteria cells 7 . Furthermore, the attachment and biofilm formation are largely mediated by pili, encoded by a csu operon cluster genes, that form a pilus-like pack structure in A. baumannii. Among csu operon, it is found that the csuE gene (Chaperon-Usher pilus) plays an important role in biofilm assemble in A. baumannii. Similarly, it was reported that a partial contribution in biofilm formation is mediated by the outer membrane protein A (OmpA), which enhanced the ability of A. baumannii to adhere to biotic surfaces such as epithelial cells and lung epithelial cells and, may induce apoptosis in epithelial cells of human 8, 9 . Several recent studies have shown that the clinical isolates of A. baumannii have a robust system to persistence on biotic surfaces; this is attributed mostly to the presence and expression of blaPER-1 genes which enhance and increase the ability of these isolates to invade lung epithelial cells 10 . Moreover, antimicrobial sensitivity can be affected by several biofilm-associated genes, and thus may refer to a correlation between biofilm formation and drug resistance 11 . However, this correlation is still unconfirmed, in recent study, the correlation between antibiotic sensitivity and biofilm formation with the associated genes are significant 12 .
Nowadays, we required a new strategy to combat the increasing problems of A. baumannii drug resistance. Low Frequency Ultrasound (LFU) is one of the safest and promising physical approaches 13 . Ultrasound has been used in research and diagnosis for decades, with frequency of 20 kHz or higher. The LFU is also known as a highpower ultrasound and has a frequency ranging from 20 to 100 kHz 14 . The effect of ultrasound on microorganisms is mediated by mechanical forces, such as shock waves, shear forces, and microjets. The creation and collapse of microbubbles in a liquid medium increase the permeability of biofilm and accelerate the penetration of antibiotics, and this cavitation is assumed to be the major mechanism behind ultrasound's bactericidal effects 15. The objective of this study is to investigate the potential effect of LFU on biofilm activity of clinical isolated A.baumannii, and the correlation between antibiotic resistance and biofilm formation of integron-harboring isolates.

Methods: Specimen Collection and Identification.
A total of 100 specimens (Urine, blood, sputum, fluids, burns and wounds) were collected from different hospitals in Baghdad, the collected samples were isolated from sputum, swab, blood, urine, and fluid included cerebrospinal fluid (CSF). A total of sixty six 66 samples were identified as A. baumannii using the morphological assays, Vitek and Epi20E. The isolates were routinely cultured on MackonCey agar plates under aerobic conditions at 37 ̊ C. All isolates were stored at -80 ̊ C for further use. In this study, A. baumannii ATCC17904 was used as a standard strains.

Antibiotic Susceptibility Test of A. baumannii
Antibiotic susceptibility pattern of A. baumannii isolates were detected using disc diffusion methods by Kirby-Bauer and based on CLSI guidelines 16 . Eighteen antimicrobial agents from different classes were used to determine the sensitivity test including ampicillin-sulbactam (SAM), amikacin (AK), ceftriaxone(CTX), ceftazidime (CAZ), cefotaxime (CFT), ciprofloxacin (CIP), cefepime (CPM), gentamicin (GM), levofloxacin(LVE), Iemipenem(IMI), meropenem (MEM), piperacillin (PIR), piperacillin-Tazobactam (PTZ), trimethoprim-sulfamethoxazole (TS), tetracycline (T)Doxycycline(DXT), ticarcillin-K clavulanate (TIM), and tobramycin (TOB). A. baumannii isolates (adjusted to 0.5 MacCferland) were spread out on Muller-Hinton agar plates (MHA) (Merck, Germany), and each antibiotic disc was placed on the plates. All plates were incubated at 37 ℃ for 24 hours. Inhibition zone was measured by calculating the diameter of hollow zones around each disc. According to CLSI guidelines, the antimicrobial efficiency against the strain in vitro is expressed as "susceptible" or "intermediate", and the strain that known as resistant or "non-susceptible" means that it is "not inhibited "in vitro by a concentration of the antibiotic disc. MIC was determined by Vitek 2 compact system (AST222 Cards).

A .baumannii Biofilm Formation Assay
In order to examine the ability of A. baumannii to form biofilm, microtiter plate assay was used 17 . Briefly, an overnight culture of A .baumannii was prepared in fresh Luria Bertani (LB) broth (OD600 of 0.5) as previously mentioned 2,18 , and incubated at 37 °C for 24 h. A 200 µl of diluted bacteria 1:50 was inoculated in 96 wells plate and incubated over night at 37℃ for 24 h. The microtitre plates were washed three times with 1 X phosphate buffered saline (PBS). The plates were fixed by methanol 99 %, and 200μl of Crystal violet 1% was added for 30 min, and left to dry at room temperature for 15 min. The microplates were washed with PBS, and all reads were recorded at ODc 570 nm using ELISA reader. The assays were done in triplicate 19 . The adhesion ability of isolates was organized into four groups. The mean optical density of the negative control (contained broth only) was deemed as the cut-off optical density (ODc). The isolates were grouped as follows: -If OD ≤ ODc, the isolate is non-biofilm producer -If ODc < OD ≤ 2×ODc, the isolate is weakly biofilm producer.

Low Frequency Ultrasound
To unequivocally determine the efficiency in the use of low frequency ultrasound (LFU) to inhibit the biofilm formation in clinical isolates A. baumannii, overnight culture of bacterial isolate was inoculated in 100 ml nutrient broth. The bacterial suspension was transferred to 250 ml conical flask and placed in a shaker incubator (200 rpm) at 37℃ for 24 h. The suspension was centrifuged at 7000 rpm for 10 min. The supernatant was discarded and the pellet was resuspended with sterile distilled water. The bacterial suspension was exposed to different intensities of 20KH 5Amp, 15Amp, 50Amp and several time intervals zero, 5, 10, 15, and 30 min, time zero is considered as OD positive control. To avoid the potential heating resulted from each exposure, temperature was continuously measured and the suspension was cooled in the ice box when it requires 20 .
To precisely assess the anti-biofilm activity of LFU, the OD600 of bacterial suspension from each exposure was measured, and the suspension was adjusted to 1×10 5 . The suspension was further diluted to 1:50 in LB broth, and about 200 µl of suspension was inoculated in polystyrene microtiter plates, followed by incubation at 37℃ for 72 h. The anti-biofilm was measured according to the following equation: Biofilm inhibition =OD (positive control -OD (treatment) / OD (positive control) ×100%

Anti-biofilm Formation Assay
Anti-biofilm formation assay was performed using the honey as a chemical compound against the clinical isolates of A. baumannii. Briefly, a loopfull of overnight bacterial growth of A. baumanni was inoculated in fresh LB broth and incubated at 37 ̊ C for 24 h. The culture was then diluted to 1:50 with LB broth and nine different concentrations of honey were added 10, 20, 30, 40, 50, 60, 70, 80, 90%, and incubated at 37 ̊ C for 24 h. Biofilm formation assay was performed as previously described in 2.3 20 .

DNA Extraction and Manipulation
DNA has been extracted according to Genomic kit's instruction procedure; the procedure has been set to isolates from each group of strong and moderate biofilm formation in addition to the standard strain of A. baumannii ATCC 17904. To determine the DNA concentration and purity, the Nano drop system has been used, and the purity has been measured depending on the following equation: DNA purity = OD260 / OD280 The concentration of DNA was calculated as: DNA concentration (μg ml -1 ) = OD260 nm *50* dilution factor 21 .

PCR for Biofilm related genes and Integrons (bap, csuE, Int1, Int2)
The PCR reaction of genes of interest (bap, csuE, Int1and Int2) was performed in a 25µl reaction mixture containing 12.5 µl of Green Master Mix, 1 µl (10 pmol µl -1 ) of forward and reverse primers listed in table 1. Genomic DNA (2 µl) was added, and the volume was completed to a final volume of 25 µl by nuclease free water. PCR conditions were set up according to annealing temperature of each primer as indicated in table 2. PCR products were resolved on 1 % agarose gel, and visualized by using UV transilluminator, and the image was captured by Gel documentation (Witeg Labortechnik GmbH, Germany).

Statistical analyses
Statistical analyses were done using oneway ANOVA and Tukey's post hoc correction. To make of multiple comparisons, all stated P values were modified. Pearson's correlation analysis was used to examine the relationship between the phenotypes. P values less than 0.05 were deemed as significance. The JASP package was used. Values of ≤0.05 were considered as significance. All data analyzed in this work were derived from at least three biological replicates.  Results:

Antimicrobial Sensitivity Profile of Clinical Isolated A. bumannii
The antibiotic susceptibility pattern of A. baumannii isolates was examined by disc diffusion assay, and the results showed that the majority of clinical isolated A .baumanni 95.5 % were resistance to ceftriaxone, ceftazidim, and ticarcillin-K clavulanate, and about 92.4 %, 91 % were resistance to cefepime and ciprofloxacin respectively, whereas, Doxycycline exhibits the lowest activity against isolates 63.6% Fig. 1, S2, S3: supplementary file.
Notably, A. baumannii isolates from fluid and swabs were resistant to most antibiotics (94.22%) and (93.9%) respectively, whereas, isolates in urine, sputum and blood were less resistant 86.12 %, 84.76 %, 81.84 % respectively. The high resistance patterns of A. baumannii isolates in fluid and swabs may be due to several resistance mechanisms mediated by related genes, resulted from the extreme using of antimicrobial agents. On the other hand, current study revealed that the increase in resistance of A. baumannii clinical isolates to different antimicrobial agents is diverse within the sources.

Determination of Minimal Inhibitory Concentration (MIC)
Minimal inhibitory concentration is defined when the value of antimicrobial agents was equal to or higher than the breakpoints, which means that the bacteria or pathogens were resistant to the antibiotics used. In the current study, the MIC was determined by Vitek 2 compact system (AST222 Cards), and the rates of resistance to the antibiotics are shown in Table 3. The results showed that most A. baumannii isolates were sensitive to colistin except for one isolate. The MIC value to imipenem was (>=16µg ml -1 ), whereas, 23 , determine the lowest MIC value to imipenem as ranged from 8 -64 µg ml -1 . It is clear that the increase in the initial breakpoint of MIC means that the antibiotic does not affect A. baumannii at a concentration lower than 16 µg ml -1 . Moreover, the reduced susceptibility to imipenem in clinical and nonclinical isolates to 80.4%-83.3% and the MIC ranged from 8-64 µg ml -1 24 .  baumannii ATCC17904 forms weak biofilm. From the above results, it is obvious that strains isolated from swab and sputum form strong biofilm than other isolates (blood, fluid, and urine) P<0.001. Most notably, there was a statistical significant difference between antibiotic resistances and biofilm formation including (strong, moderate, weak and non-biofilm) isolates at a statistical level P<0.0001 Fig. 4, S5: Supplemented file.

Biofilm Inhibition using of LFU
The potential activity of LFU to reduce the ability of A. baumanni isolates to form biofilm was evaluated. The analysis of biofilm inhibition revealed that the biofilm ability was decreased from 44.3% to 50.7% at 5 min exposure and under different amplitudes 5, 15, 50 AMP/ 20KH. Fig. 5, whereas, after 10 min exposure, the inhibition percentage of biofilm is increased remarkably from 51.7 to 57.9 % under similar intensities. However, there is no significant reduction in biofilm production after 15 min 58.7 to 60.7%, and 30 min 58.7% to 62.9%. ̋ S6: supplementary file . On the other hand, ODc 600 nm was measured after each exposure to LFU, and the results showed that there is an obvious decrease in the density of all treated isolates (Data not Shown). The impact of LFU on antibiotic susceptibility profile of A. baumannii was determined. The results indicate that there is no obvious effect on A. baumannii susceptibility pattern (Data not Shown).
The probability to inhibit the biofilm formation by clinically isolated A. baumannii chemically was also investigated ̋ S7: Supplementary file . The 5-hydroxymethylfurfural; an organic compound presents in the honey; is annotated to be able to inhibit biofilms formation of A. baumannii. The mechanism mediated by 5hydroxymethylfurfural is unknown, but it was reported once that this compound is toxic for human

Detection of Biofilm -Related genes (bap, csuE)
One of the objectives of the present study is to detect the presence of common biofilm related genes in all A. baumannii isolates. The majority of 15 isolates including: strong, moderate and non-biofilm producer were subjected to investigate the presence of the main biofilm-related genes (bap, csuE), and whether the resistance pattern is biofilm dependent. The genes of interest were PCR amplified using primer pairs listed previously in Table 1. The PCR products were electrophoresed and visualized under the UV light. Interestingly, the results showed that the prevalence of the bap gene 650 bp was 93.3%, whereas the bap gene was absent in one strong biofilm forming isolate 6.7% Fig. 6A. On the other hand, the prevalence of chaperone-usher pili assembly system gene csuE 103 bp were 86.6% in A. baumannii is.olates, and 13.4% of isolates were lack the csuE gene Fig. 6 B.
In order to unequivocally determine the correlation between antibiotic resistance and biofilm formation ability of A. baumannii, integron genes (intl-1 and intl -2) were detected in 15 selected clinical isolates of A. baumannii. The amplified genes were analyzed with 1.5 % agarose gel, and the results showed that the prevalence of the intl -1 200 bp and intl-2 700 bp was 87.5% and 13.3% respectively, whereas, about 12.5% and 86.6% of selected isolates were lack the intl-1 and intl-2 genes respectively Fig. 6 C, D.

Figure 5. Biofilm Inhibition percentage of Low Frequency Ultrasound against A. baumannii clinical isolates.
A culture of A. baumannii isolates was exposed to different amplitudes of LFU, the isolates were subjected to biofilm assays and the inhibition percentage was calculated. A: represents the inhibition percentage at 5AMP; B: inhibition percentage at 15AMP; C: inhibition percentage at 50AMP. The experiments were done in triplicates. of (bap, csuE, intI-1, and intI-2)

Discussion:
In the recent years, A. baumannii have been reported as nosocomial pathogens due to their prevalence in the hospitalized patients. Statistical analyses of A. baumannii isolated based on the source of isolation indicate that the prevalence in swabs (burns and wounds) 33.3%, sputum 28.8%, and blood 22.7% was higher than that in fluid 9.1%, and urine 6.1%. Locally, it was reported that the majority of A. baumannii were isolated from sputum 61%, wounds 19%, blood 9%, urine 3%, and other specimens 8 % included pus, synovial fluid, and ascetic fluid 25 . Furthermore, it was recorded that the majority of A. baumannii strains were isolated from sputum, wound, swabs, and urine 26 . Our findings were agreed with 27 who reported that A. baumannii was isolated from burn skin and wound. A. baumannii have several adhesion factors facilitate the colonization and persistence in the respiratory system and mucous cells.
The prevalence of A. baumannii in Baghdad hospitals became the main issues and raise a remarkable interest that required serious steps to turning off the rapid spread of this pathogen and decrease the mortality and morbidity rates. In the current research, A. baumannii isolates were resistant to mainly all antimicrobial agents used 86.7%. The resistance to ciprofloxacin 90.9 %, imipenem 83.3%, and gentamicin 89.4% is similar to 28 , who showed that the resistance to Ciprofloxacin was 95%, meropenem 74.2%, and Gentamicin 35%.
Moreover, it was reported that the majority of A. baumannii isolates 92% were resistant to Amikacin, and 83.3% were resistant to tetracycline 29 . Moreover, 84.8% of A. baumannii isolates were resistant to levofloxacin, however, it was mentioned that 91% of pathogenic isolates were resistant to levofloxacin 30 .
In addition, of the 66 clinical A. baumannii isolates evaluated, 1.5 % were qualified as PDR, 72.7 % were designed as XDR and 16.7 %, were qualified as MDR, whereas, 85.6 % of A. baumannii isolates were designed as MDR, and 14.4% were qualified as XDR about 31 . The resistance phenotype of Gram negative bacteria in urine is diverse, among all isolates in urine 35, 62.8 % were qualified as MDR, and 28.5 % were qualified as XDR. However, they all are considered as biofilm producers 32 .
The clinical isolates of A. baumannii exert different patterns of resistance against antibiotics, 95.5% were resistance towards ceftazidime, ceftriaxone and CTX, and 90.9 % were resistance to ciprofloxacin and piperacillin. It was reported by Mekkey et al., 2020 33 , that 100% of A. baumannii were resistance to ceftazidime, 83 % were resistant to meropenem, and 98 % were resistant to ciprofloxacin. Most notably, all A. baumannii strains isolated from different sources display various resistance profiles to most antibiotics, all A. baumannii isolates were not susceptible to carbencillin, but 50 % were resistance to ampicillinsulbactum 34 .
In addition, the resistance to ampicillin-sulbactam was increased 65%, due to the prevalent use of antimicrobial agents in Baghdad hospitals. The resistance pattern of A. baumannii strains was varied in AL-yarmok Teaching Hospital, as 65% were resistance to piperacillin, 100 % were resistance to cefotaxime, 65% were resistance to ceftazidime, 100 % were resistance to cephalexin, 65% were resistance to ceftriaxone, 50% were resistance to imipenem 35 . Additionally, the majority of A. baumannii 99% were resistant to ciprofloxacin, 98% were resistance to ceftazidime, meropenem, and imipenem, 77% were not sensitive to gentamicin, and 48% were resistance to amikacin 36 . Our results showed that most of A. baumannii isolates 98.5% were highly susceptible to colistin, as it mainly targets the LPS components, and attenuates the colonization of A. baumannii in lung epithelial cells 37 .
Based on our results, of the 66 clinical A. baumannii isolates; 11, 16.7% were designed as MDR, 48, 72.7% were qualified as XDR, 1.5% was qualified as PDR, and 6, 9.1% were susceptible to the most antibiotics. The prevalence of XDR isolates is attributed to the extensive use of antibiotics in Iraqi hospitals 38 .
The ability of A. baumannii isolates to form biofilm promotes the persistence in different environments, and increasing the risk of healthcareassociated infections. This study assesses the biofilm-production capacity of the clinical isolates A. baumannii, and the potential correlation between biofilm formation and susceptibility to antibiotics. Most notably, there were significant statistical differences between biofilm formation ability and antibiotic resistance at level p<=0.01, indicating for a positive correlation between biofilm patterns and antibiotic resistance 3 . The current study revealed that most A. baumannii isolates 72.7 % were able to form biofilm, but only 20% isolates were able to produce strong biofilm, because the link between biofilm forming capacity and pathogenic resistance to antibiotics is currently unorganized, and the outcome between studies was even contrast 39,40 . However, a positive correlation between bacterial antibiotic resistance and biofilm formation was mentioned 41 in contrast with finding of our result which, indicating that these pathogens were not depending on antibiotic resistance for survival, as the non-biofilm forming isolates were resistance to antibiotics as well. Our study indicated that the antimicrobial resistance is not correlated with the biofilm forming ability, and antibiotic resistance is one of the main survival mechanisms for A. baumannii, in addition to biofilm forming capacity.
Furthermore, most of 75% A. baumannii isolates were able to form biofilm, of which 43% isolates were able to form strong biofilm, and 32% isolates were formed moderate biofilm, while, 25% were produced weak and nonbiofilm producers. It was notably that the biofilm formation ability decreases the effect of antimicrobial agents, and alternative strategies of treatment are seriously needed, there for, dissecting of the virulence factors in pathogenic bacterial isolates is urgent, and important with the use of most authoritative methods 42 .
Because the ability of pathogens to form biofilm is not correlated with the resistance to antimicrobial agents, there are significant challenges needed to inhibit the biofilm formation of A. baumannii isolates. In this study, among 66 of A. baumannii isolates, three isolates evaluated as strong biofilm formers, were exposed to LFU. The results investigate that the sonication of bacterial suspension at continues frequency 20 KHz, stimulates the initial decrease in the number of bacterial cells, and the number of cells was continuously decreased after 15 min exposure. Subsequently, there was a gradual and continuous decrease in the number of bacterial cells after 30 min exposure 43 . When a bacterial suspension is exposed to low frequency ultrasound, a large bubble cavitation will form and fall, this will generate a high level of energy, and in contrast, high frequency ultrasound generates shorter time for cavitation bubbles to form with less energy, ultimately, leads to collapse 44 . This indicates that low frequency ultrasound 20 KHz is might be a good approach to significantly reduce the bacterial suspension. In addition, there are many factors affect the sensitivity of bacteria to sonication such as; the composition of medium, the movement of sound, distribution of power and viscosity 45 .
However, the using of LFU will temporarily decrease the biofilm formation of A. baumannii, there are many factors enhance the efficiency of LFU such as; power intensity, environmental conditions, frequency, time of irradation, and duty cycle. Low frequancy ultrasound is a promising assay to decrease the biofilm formation ability due to many reasons; the probe directivity, and remedation efficiency, both target the deep tissues without damaging the tissue. Furthermore, the low frequency ultrasound approach may release extra oxygen and nutrition into the biofilm, which accelerates the bacterial growth and retrieves the antimicrobial susceptibility to antibiotics 46 .
It was demonstrated that the continuous wave of ultrasound at a frequency of 70 KHz, and power density 10mW/cm 2 are efficient to improve the bactericidal effect of antibiotics more than low intensity 1mW/cm 2 , and the pulsed wave intensity is effective as a continuous ultrasound with identical density 21 .
On the other hand, the chemical components in honey play potential roles as antimicrobial drugs against A. baumannii isolates, the activity is due to several factors; high osmatic effect, non-hydrogen peroxide, phytochemical components that involved in the controlling of bacterial colonization, and subsequently disruption of biofilm 47 . Additionally, it was suggested that the high concentration of sugar and high acidity in honey reduce the ability of pathogens to form biofilm. Our study revealed that there was a significant invert correlation between the high concentration of honey and biofilm inhibition rate 48,49 .

Biofilm-related genes detection
The ability of A. baumannii isolates to form biofilm is induced by a complex of genes that involved in biofilm maturation and completion. In our study, the bap gene was screened out in 15 isolates. It was reported that the frequency of bap gene in the isolates were 92%, and there was a strong correlation between the bap gene and the biofilm production 50 . The bap gene encodes for Bap protein, is one of the major virulence factors and plays a substantial role for adherence to the epithelial cells and maturation of biofilms. In addition, another study 51 found that 89% of MDR A. baumannii have bap gene and suggested that there was strong correlation between biofilm formation and biofilm gene in the multi -drug resistance A.baumannii. Furthermore, the prevalence of bap gene in 30 clinical A. baumannii isolates was 76.6%. The prevalence of bap genes in the strong biofilm forming isolates is clearly explained the ability of isolates to form biofilm, and this may be attributed to the influence of biofilm associated protein (Bap) in the biofilm maturation 50 .Our findings showed a significant correlation between bap gene and biofilm formation ability. Our findings are similar to 66, who demonstrated a strong correlation between biofilm formation and bap genes. Furthermore, bap gene was detected in 48.43% clinical A. baumannii isolates 52 .
According to our results, of the 15 evaluated A. baumannii isolates, the prevalence of csuE was 86.6%. Similarly, the prevalence of csuE gene in clinical isolates A. baumannii was 93.2% 53,54 . Moreover, it was recorded that the csuE gene was predominance in all evaluated A. baumannii isolates 100% 25 . Based on the above findings, there was a strong correlation between biofilm, antibiotic resistance, and biofilm related genes (bap, csuE), as the bacterial pathogens in the biofilm community are able to tolerate the antimicrobial agents 100 to 1000 folds more than the plankton community 55 . Furthermore, the formation of pili is fundamental for the initial steps in biofilm formation. The results of the current study revealed the prevalence of (bap and csuE) genes in strong, moderate, and weak biofilm producers, these results proposed that the expression level of these genes play a key role in the determination of biofilm formation capacity 56 .

Integrons
In our study, the prevalence of integrons (Intl-1and intl-2) genes in A. baumannii isolates were 87.5% and 13.3% respectively. Many mentioned that several factors involved in the distribution of integrons within clinical isolates in particular, for example; the wrong uses of antibiotics for treatment, overexpression of gene cassettes included integrons class -1, integrons capacity to accept a new gene cassette, and disability to build a national surveillance system in the medical centers as a part of the national managements for antimicrobial resistance mechanisms. Because of the Intl-1 gene is transferred by a common genetic elements such as; conjugative, plasmids, and transposons, suggested to be the main reasons for the significant increase in the emergence of MDR A. baumannii in Iraqi hospitals 57,58 .
The study of 59 reported that the prevalence of intl-1 and intl-2 genes among the clinically isolates A. baumannii were 63.9% and 78.2% respectively. However, many studies have been conducted to investigate the presence of integron class-I worldwide. Most notably that, the prevalence of integron class-1 is variable, due to the geographical distribution of A. baumannii, the source of infection, or the source of isolation 60 .
Furthermore, the results clearly showed that there was a strong correlation between the prevalence of intl-1 gene and antimicrobial resistant patterns, and with the biofilm formation capacity. These correlations explain the significant role of integrons in antibiotic resistance and thereby in the epidemic attitude of A. baumannii 61 . Other study 62 found that the prevalence of integron class-1 and integron class-2 were 70.77 % and 26.15% respectively. Integrons; are transposons (like genetic elements), have a conserved region, and encodes for antibiotic resistance determinants and have extra ability for chromosomal integration in bacteria 58 .
Many classes of integrons have been identified, and the most common classes are integrons class-1 and class-2 in MDR A. baumannii strains 66. According to the previous study, integron class-1 is the most important gene in the distribution of MDR A. baumannii 62 .
Our study demonstrated that the integron class-1 was predominant in the (XDR) and (MDR) A. baumannii isolates, and there was a correlation between integron class-1 and the resistance gene cassettes.

Conclusion:
The predominance of A. baumannii in Baghdad hospitals is particularly in Gazii AL-Hariry Hospital, and burn hospital increasing day by day, and the increase in antibiotics resistance is crucial. A. baumannii clinical isolates exhibit high resistance ability to cephalosporins, whereas the isolates were sensitive toward doxycycline, suggesting that doxycycline is effective against A. baumannii. In addition, A. baumannii is well known for its ability to form biofilm on biotic and abiotic surfaces, so novel approaches are needed to attenuate and decrease biofilm formation ability. The use of green energy of LFU with low frequency ultrasound is a promising technique to inhibit biofilm production, particularly at low energy and short time. In addition, the natural components in honey reduce the biofilm production. Subsequently, they reduce the transfer of pathogen through medical equipment, and decrease the number of patients infected with A. baumannii.