Biofilm Formation in Acinetobacter Baumannii: Genotype-Phenotype Correlation

Strains of Acinetobacter baumannii are commensal and opportunistic pathogens that have emerged as problematic hospital pathogens due to its biofilm formation ability and multiple antibiotic resistances. The biofilm-associated pathogens usually exhibit dramatically decreased susceptibility to antibiotics. This study was aimed to investigate the correlation of biofilm-forming ability, antibiotic resistance and biofilm-related genes of 154 A. baumannii isolates which were collected from a teaching hospital in Taiwan. Biofilm-forming ability of the isolates was evaluated by crystal violet staining and observed by scanning electron microscopy. Antibiotic susceptibility was determined by disc diffusion method and minimum inhibitory concentration; the biofilm-related genes were screened by polymerase chain reaction. Results showed that among the 154 tested isolates, 15.6% of the clinical isolates were weak biofilm producers, while 32.5% and 45.4% of them possessed moderate and strong biofilm formation ability, respectively. The experimental results revealed that the multiple drug resistant isolates usually provided a higher biofilm formation. The prevalence of biofilm related genes including bap, blaPER-1, csuE and ompA among the isolated strains was 79.2%, 38.3%, 91.6%, and 68.8%, respectively. The results indicated that the antibiotic resistance, the formation of biofilm and the related genes were significantly correlated. The results of this study can effectively help to understand the antibiotic resistant mechanism and provides the valuable information to the screening, identification, diagnosis, treatment and control of clinical antibiotic-resistant pathogens.


Introduction
Acinetobacter baumannii is an important nosocomial pathogen that is responsible for a wide range of human infections [1,2]. Recently, the rapid development of multiple antibiotic resistance of A. baumannii has caused a serious problem for public health. The ability of biofilm formation contributes to Acinetobacter easily survive and transfer in the hospital environment, such as attached to various biotic and abiotic surfaces, e.g., vascular catheters, cerebrospinal fluid shunts or Foleys catheter [3,4]. Biofilms are assemblages of microorganisms, encased in a matrix, that function as a cooperative consortium to provide a protected mode for microorganisms and enhance resistance to various antibiotics [5]. Biofilm formation is a complex process employing many factors that include the aggregation substance, adhesion of collagen, expression of pili, and iron acquisition [6].
Among the several factors, the biofilm-associated protein encoded by the bap gene plays an important role in intercellular adhesion, accumulation of bacterial cells, and establishment of 2 of 12 biofilm [7,8]. In the literature reports, the presence and expression of the bla PER-1 gene has been identified to encourage the clinical isolates of A. baumannii to form biofilm and adhere to respiratory epithelial cells [9][10][11]. The report extends previous observations by showing that the outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on the plastic surface [10]. The ability of A. baumannii to form biofilms is also largely dependent on pili, which mediate attachment and biofilm formation. The genes are clustered together in the form of a csu operon, the products of which form a pilus-like bundle structure in A. baumannii [12]. Hence, the csuE gene also plays a major role in A. baumannii biofilm formation [13]. The bacterial and fungal biofilm formation has been suggested to decrease the diffusion of drugs through the bacterial and fungal cells and cause the persistence of clinical isolates under harsh environments with multidrug resistance [14][15][16][17].
However, it is currently unclear whether there is a quantitative correlation between biofilm formation and antibiotic resistance. In this study, 154 clinical A. baumannii isolates were investigated for their antibiotic susceptibility profile, biofilm formation and the biofilm related genes; we also analyzed the relationship between their phenotypes and genotypes.
The objective of this study was to determine the correlation between the ability of biofilm formation with distribution of biofilm related genes and antibiotic resistance phenotypes in the clinical isolates of Acinetobacter baumannii.
Molecules 2019, 24, x FOR PEER REVIEW 2 of 12 [7,8]. In the literature reports, the presence and expression of the blaPER-1 gene has been identified to encourage the clinical isolates of A. baumannii to form biofilm and adhere to respiratory epithelial cells [9][10][11]. The report extends previous observations by showing that the outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on the plastic surface [10]. The ability of A. baumannii to form biofilms is also largely dependent on pili, which mediate attachment and biofilm formation. The genes are clustered together in the form of a csu operon, the products of which form a pilus-like bundle structure in A. baumannii [12]. Hence, the csuE gene also plays a major role in A. baumannii biofilm formation [13]. The bacterial and fungal biofilm formation has been suggested to decrease the diffusion of drugs through the bacterial and fungal cells and cause the persistence of clinical isolates under harsh environments with multidrug resistance [14][15][16][17]. However, it is currently unclear whether there is a quantitative correlation between biofilm formation and antibiotic resistance. In this study, 154 clinical A. baumannii isolates were investigated for their antibiotic susceptibility profile, biofilm formation and the biofilm related genes; we also analyzed the relationship between their phenotypes and genotypes.
The objective of this study was to determine the correlation between the ability of biofilm formation with distribution of biofilm related genes and antibiotic resistance phenotypes in the clinical isolates of Acinetobacter baumannii.

Relationship between Antibiotic Susceptibility and Biofilm Formation
The correlation between biofilm formation and resistance to the 11 antimicrobial agents in A. baumannii was analyzed using the Wilcoxon rank-sum test [19]. Antibiotic resistance was determined for the 11 agents covering the six antimicrobial categories, namely aminoglycosides, cephems, carbapenems, penicillins, folate pathway inhibitors, and tetracyclines. Among the 154 test isolates, only 6.4% were not biofilm producers, 15.6% were weak biofilm formers, 32.4% (50 isolates) were moderate biofilm formers, and 45.4% (70 isolates) were strong biofilm formers ( Table 2). To determine whether biofilm formation is correlated with any particular antibiotic resistance, biofilm formers with different resistance profiles for the 11 antibiotics were compared. As shown in Figure 2, the results revealed that for the ticarcillin ( Figure 2B), ceftazidime ( Figure 2C), gentamicin ( Figure 2E), and piperacillin ( Figure 2G) antibiotics, the resistant isolates tended to form stronger biofilms than the intermediate isolates (p = 0.018, 0.003, 0.003, and 0.033, respectively). For the ticarcillin ( Figure 2B), imipenem ( Figure 2F), and sulfamethoxazole-trimethoprim ( Figure 2K) antibiotics, the susceptible isolates tended to form weaker biofilms than the intermediate isolates (p < 0.001, 0.017, and 0.020, respectively). In addition, the isolates with resistance to amikacin (Figure 2A), ticarcillin ( Figure 2B), and sulfamethoxazole-trimethoprim ( Figure 2K) exhibited stronger biofilm formation than the susceptible isolates (p = 0.004, p < 0.001, and p = 0.007, respectively). The results indicate a positive correlation between biofilm formation capacity and resistance to amikacin, ticarcillin, ceftazidime, gentamicin, piperacillin, imipenem, and sulfamethoxazole-trimethoprim antibiotics. For four out of the 11 antibiotics tested (cephalexin, Figure 2D; streptomycin, Figure 2H; tetracycline, Figure 2I and carbenicillin, Figure 2J), no significant difference in biofilm formation was observed between susceptible and resistant isolates (p > 0.05). Due to the substantial differences in sample size, only one isolate was susceptible with immediate resistance to cephalexin, six isolates had immediate resistance to tetracycline, and six isolates were susceptible to carbenicillin. The results might not be confirmed by statistical analysis.
To determine whether biofilm formation is correlated with any particular antibiotic resistance, biofilm formers with different resistance profiles for the 11 antibiotics were compared. As shown in Figure 2, the results revealed that for the ticarcillin ( Figure 2B), ceftazidime ( Figure 2C), gentamicin ( Figure 2E), and piperacillin ( Figure 2G) antibiotics, the resistant isolates tended to form stronger biofilms than the intermediate isolates (p = 0.018, 0.003, 0.003, and 0.033, respectively). For the ticarcillin ( Figure 2B), imipenem ( Figure 2F), and sulfamethoxazole-trimethoprim ( Figure 2K) antibiotics, the susceptible isolates tended to form weaker biofilms than the intermediate isolates (p < 0.001, 0.017, and 0.020, respectively). In addition, the isolates with resistance to amikacin (Figure 2A), ticarcillin ( Figure 2B), and sulfamethoxazole-trimethoprim ( Figure 2K) exhibited stronger biofilm formation than the susceptible isolates (p = 0.004, p < 0.001, and p = 0.007, respectively). The results indicate a positive correlation between biofilm formation capacity and resistance to amikacin, ticarcillin, ceftazidime, gentamicin, piperacillin, imipenem, and sulfamethoxazole-trimethoprim antibiotics. For four out of the 11 antibiotics tested (cephalexin, Figure 2D; streptomycin, Figure 2H; tetracycline, Figure 2I and carbenicillin, Figure 2J), no significant difference in biofilm formation was observed between susceptible and resistant isolates (p > 0.05). Due to the substantial differences in sample size, only one isolate was susceptible with immediate resistance to cephalexin, six isolates had immediate resistance to tetracycline, and six isolates were susceptible to carbenicillin. The results might not be confirmed by statistical analysis.

Microscopic Analysis of Biofilms Formation Ability
Biofilm formation on the minimum biofilm eliminating concentration (MBEC) device was observed using scanning electron microscopy (SEM). The SEM analysis revealed that in the moderate-biofilm-forming strains, only a few of the cells were clustered together, whereas in the strong-biofilm-forming strains, large groups of conglomerate cells were found (Figure 3). To analyze the effects of antibiotics on biofilm formation, the isolates were treated with different doses of imipenem and different growing times. SEM images indicated that the biofilm formation is related to treatment time and antibiotics dosage. As shown in Figure 4, the biofilm was clearly inhibited at a higher concentration of imipenem (64 µg/mL) and after longer treatment (8 hr).

Microscopic Analysis of Biofilms Formation Ability
Biofilm formation on the minimum biofilm eliminating concentration (MBEC) device was observed using scanning electron microscopy (SEM). The SEM analysis revealed that in the moderate-biofilm-forming strains, only a few of the cells were clustered together, whereas in the strong-biofilm-forming strains, large groups of conglomerate cells were found (Figure 3). To analyze the effects of antibiotics on biofilm formation, the isolates were treated with different doses of imipenem and different growing times. SEM images indicated that the biofilm formation is related to treatment time and antibiotics dosage. As shown in Figure 4, the biofilm was clearly inhibited at a higher concentration of imipenem (64 µg/mL) and after longer treatment (8 hr). Molecules 2019, 24, x FOR PEER REVIEW 6 of 12 A B

Discussion
Acinetobacter baumannii, recently as an increasingly common pathogen, is closely associated with hospital acquired infection [1,2]. Many studies have found that the strong survival ability of A.

Discussion
Acinetobacter baumannii, recently as an increasingly common pathogen, is closely associated with hospital acquired infection [1,2]. Many studies have found that the strong survival ability of A.

Discussion
Acinetobacter baumannii, recently as an increasingly common pathogen, is closely associated with hospital acquired infection [1,2]. Many studies have found that the strong survival ability of A. baumannii in strict environments and highly resistant to various antibiotics is due to biofilm formation [3][4][5][6]. The present study investigated relationships among antibiotic resistance, biofilm formation, and the related genes in the clinical isolates of A. baumannii. A phenotype profile was compared with biofilm formation and antibiotic resistance, and we observed that antibiotic resistance was highly associated with the biofilm formation capacities. Some of the antibiotic resistant strains had higher biofilm formation capacities under certain antibiotics; for example, penicillin-resistant strains exhibited a greater biofilm formation capacity [20]. That might be due to a constitute stress such as antibiotics that will enhance for induced gene regulation and offer fitness advantages for resistant strains, resulting in biofilm formation [21]. The results suggested that penicillin resistance had a positive correlation with biofilm formation capacity.
Biofilm formation and antibiotic resistance levels may vary among sites and the key factors responsible for this resistance may differ. Regarding resistance, the primary evidence indicates that conventional mechanisms cannot explain the high resistance to antibacterial agents associated with biofilms [22]. Several mechanisms considered key factors in the high resistance of biofilms have been explored: (a) limited diffusion, (b) enzyme-caused neutralizations, (c) heterogeneous function, (d) slow growth rate, (e) persistent (nondividing) cells, and (f) biofilm phenotype adaptive mechanisms [22,23].
Agar-based antibacterial susceptibility testing, such as the disk diffusion method, has a lower cost and less labor compared with the broth dilution method. In addition, the disk diffusion assay only provides a zone of inhibition and does not generate a minimum inhibitory concentration (MIC) for each antibiotics tested. Thus, according to the results obtained from the disk diffusion test, we selected 75 A. baumannii isolates for the MIC determination by the broth dilution assay. Some recent studies have reported that exposure of strains to MICs of certain antibiotics promotes biofilm formation, indicating that biofilms tend to be more robust when antibiotic resistance is challenged [22][23][24], this is consistent with the results of the present study. In addition, our study found that the strong biofilm producers tended to be resistant against numerous antibiotics, including ticarcillin, ceftazidime, gentamicin, and piperacillin. Among these antibiotics, ticarcillin and piperacillin belong to penicillin antibiotics and their robust biofilm formation is associated with antibiotics in the penicillin class, as reported in the previous research [24]. However, in the present study, we found that resistance to aminoglycoside antibiotics was also related to biofilm formation; this has not been reported in any previous studies. We postulate that this may be because aminoglycosides are frequently ineffective against strains of A. baumannii, and thus combinations of aminoglycosides and carbapenems are often applied to yield synergistic effects for treatment of infected patients in hospitals [25]. Therefore, the positive correlation between aminoglycoside resistance and biofilm formation could be due to the synergistic effects of both antibiotics.
Although no studies have reported a relationship between aminoglycoside resistance and biofilm formation in A. baumannii, Hoffman et al. observed that aminoglycoside antibiotics induced biofilm formation in P. aeruginosa and Escherichia coli [26]. In P. aeruginosa, a gene, namely aminoglycoside response regulator (arr), was essential for induction and contributed to biofilm-specific aminoglycoside resistance. In the present study, based on the results of antibiotic susceptibility tests, aminoglycoside antibiotics induced bacterial biofilm formation in A. baumannii. In addition to the correlation between antibiotic resistance and biofilm formation, the relationship between biofilm formation and related genes, including bap, csuE, ompA and bla PER-1 , were evaluated in this study. The biofilm associated protein is expressed on the cell surfaces of bacteria; many of the bap gene carriers of A. baumannii exhibit biofilm production on both biotic and abiotic surfaces [7,8]. In the study, molecular analyses showed that 122 (79.2%) clinical isolates of A. baumannii harbored the bap gene. In addition, the statistical analysis revealed that the emergence of bap and biofilm formation was related to the connection. The biofilm related gene, csuE, is a member of the usher-chaperone assembly system, which mediate attachment and biofilm formation. In the present study, the csuE gene harboring strains accounted for 68.8% of the test isolates.
In 2008, Lee et al suggested that biofilm formation in A. baumannii was related to the bla PER-1 gene [11]. A. baumannii individuals harboring the extended-spectrum-resistant gene bla PER-1 formed a considerably higher biofilm formation than those that lacked bla PER-1 [27,28]. In the present study, the prevalence of the bla PER-1 gene was 38.3% in the test strains. However, one study [29] reported no relationship between biofilm formation and production of PER-1 β-lactamase. Therefore, a possible explanation for the striking characteristic of A. baumannii could be that bla PER-1 increases the adhesion of cells that carry the gene without necessarily contributing to biofilm formation.
Among the outer membrane proteins identified in A. baumannii, AbOmpA (OmpA) is the most abundant surface protein [23]. AbOmpA, acts as a porin, is required for eukaryotic cell adhesion, and partially contributes to serum resistance and biofilm formation [30]. The OmpA harboring strains accounted for 91.6% of the strains in the current study, and some of the non-biofilm-forming strains also contained the OmpA gene. However, no further evidence is available to ascertain whether OmpA induces biofilm formation.
Scanning electron microscopy (SEM) is a useful tool for investigating surface structures of biological samples [31]. In a SEM observation, A. baumannii cells were connected to one another with extracellular appendages [31]. Imipenem, a subgroup of carbapenems antibiotics, has a broad spectrum of activity against aerobic and anaerobic Gram positive as well as Gram negative bacteria. Many previous studies demonstrated that imipenem was highly effective against biofilm formation [31,32]. Thus, we used imipenem to determine the effect of antibiotics treatment on biofilm formations. In the present study, we conducted the SEM observation to determine the effect of imipenem treatment on the surface structures of biofilms grown on Minimum Biofilm Eradication Concentration (MBEC) pegs. No studies have reported the correlation between genotypes and adherence by prokaryotic cells [33]. The SEM diagrams revealed the role of imipenem on biofilm production, although the mechanism has not yet been clearly elucidated. The quantitative differences in biofilm formation among clinical isolates and their relationships with the epidemicity of strains and severity of infections have been poorly investigated, and thus such critical aspects require further study [34].
The experimental results were analyzed through statistical methods and revealed that biofilm formation is associated with the following five antibiotics: Tetracycline, sulfamethoxazole-triethoprim, gentamicin, ceftazidime, and ticarcillin. These five antibiotics, commonly used in hospitals, are categorized into five types: Tetracycline, folate pathway inhibitors, aminoglycosides, carbapenems, and penicillins. Based on the selection of antibiotics, biofilm formation by pathogens exhibits varying performance. Although not every antibiotic is associated with stronger biofilm formation, statistical analyses have revealed that biofilm formation is related to a strain's susceptibility to an antibiotic.

Bacterial Strains
A total of 154 antibiotic resistant strains of Acinetobacter baumannii were isolated from Chiayi Christian Hospital (Chiayi, Taiwan). All strains were stored at -80 • C, and bacteria were grown overnight at 37 • C on Mueller-Hinton agar (MHA). Standard strain used in this study was Acinetobacter baumannii ATCC19606.

Detection of Biofilm Related Genes
Polymerase chain reaction (PCR) assays for detection of bap, blaPER-1, csuE and ompA genes were performed by a set of primers as shown in Table 3 [29,32,35]. DNA was extracted from each isolate by genomic DNA extraction kit (Geneaid, Taiwan). PCR assays were performed using PCR Red Master Mix (AMPLIQON, Paris, France) in an ABI thermo cycler (Applied Biosystems 2720, Foster City, CA, USA). PCRs were carried out in 25 µL reaction volume and consisted of 5 µL of genomic DNA (5 ng), 12.5 µL PCR Master Mix, 2.0 U of Taq DNA polymerase, 10 mM dNTP mix at a final concentration of 0.2 mM, 50 mM MgCl 2 at a final concentration of 1.5 mM, 1 mM of each primer, 1X PCR buffer (final concentration) and 1 µL (10 pmol) of each primer. Conditions for the PCR were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 60 s, an annealing temperature for each gene (according to Table 1) for 1 min, an extension at 72°C for 45 s, followed by a final extension at 72°C for 5 min. Positive and negative controls were included in all PCR assays. Table 3. The primers used in this study for detection of biofilm related genes.

Quantitative Biofilm Formation Assay
The biofilm formation ability of A. baumannii isolates was determined by polystyrene tube assay based on the crystal violet staining method [33]. Briefly, polystyrene (12 mm × 75 mm) tubes containing 1.5 ml of Mueller-Hinton broth were inoculated with 30 µL of an overnight liquid culture with OD 600 = 0.1, and the tubes were incubated at 37 • C for 48 h. The liquid media was discarded, and the adherent cells were washed twice with phosphate-buffered saline (PBS) and stained with 0.02% of crystal violet for 10 min. The stain was eluted from the adherent cells using an ethanol solvent and vortexing for 5 min. Absorbance of the eluted solvent was measured, after diluting 10-fold with the solvent, at 580 nm using an UV visible spectrophotometer (Shishin, SH-U830, Taipei, Taiwan, ROC). The assay was done at least three times using fresh samples each time.

Microscopic Analysis of Biofilms Formation Ability
The biofilm formation ability of A. baumannii strains was visualized by scanning electron microscope (SEM) (Hitachi-S3400, Tokyo, Japan). Biofilm was formed on the minimum biofilm eliminating concentration device (MBEC™ P&G Physiology & Genetics Innovotech, Alberta, Canada). Briefly, A. baumannii suspensions (200 µL) were inoculated into each well and then incubated overnight at 37 • C. Biofilms that formed were then washed twice with PBS to remove any unattached and floating cells and were fixed with 2.5% glutaraldehyde in 0.1 M cacodylic acid (pH 7.2) at 4 • C for 24 h and post fixed with 0.1 M cacodylic acid for approximately 10 min. After incubation, the plates were washed twice with distilled water for 15 min, followed by gradual dehydration with ethanol, and air dry for a minimum of 24 h. The fixed biofilms were then coated with a layer of gold-palladium (7 nm thick) and examined with SEM (Hitachi-S3400) [36].

Statistical Analyses
The relationship between biofilm formation and antibiotic susceptibility was analyzed by Wilcoxon rank sum test. All analyses were carried out with one-way ANOVA. Categorical variables between more than two groups were tested, and P values of ≤ 0.05 indicated statistical significance.

Conclusions
In this study, the molecular genotypes and phenotypes of clinical antibiotic-resistant A. baumannii were investigated, and the correlations among antibiotic resistance, biofilm formation, and biofilm related genes were determined. Our results indicated that the ompA and bap genes influence biofilm formation and antibiotic resistance patterns based on the statistical analysis. Such mechanisms may facilitate our understanding of the relationship between biofilm production and antibiotic resistance in A. baumannii, and that of the routes of transmission of clinical isolates. The relationship between biofilm formation and antibiotic resistance may further provide information that could facilitate attempts to control drug-resistant pathogens.