A Comprehensive Study of the Relationship between the Production of β-Lactamase Enzymes and Iron/Siderophore Uptake Regulatory Genes in Clinical Isolates of Acinetobacter baumannii

Background The iron/siderophore uptake system (IUS) involved in the Acinetobacter baumannii pathogenicity. However, IUS's role in antibiotic resistance and the production of β-lactamase enzymes of A. baumannii are unclear. This study aimed to investigate the relationship between the production of β-lactamase enzymes and IUS regulatory genes in clinical isolates of A. baumannii. Methods. A. baumannii isolates were collected from clinical isolates using biochemical tests. The antibiotic resistance patterns and β-lactamase-producing strains were identified using the disk diffusion method (DDM). Also, IUS genes were detected by the polymerase chain reaction (PCR) method. Results Seventy-two (72) A. baumannii isolates were collected from a different clinical specimen. Gentamicin-resistant strains (43%) had the highest frequency, and aztreonam-resistant strains (12.5%) had the lowest frequency. Also, the distribution of AmpC and MBL producing isolates were 27.7% and 35%, respectively. Moreover, the frequencies of basD, bauA, pld, paaE, entA, feoB, hemO, and tonB genes were as follows: 12.5%, 15.2%, 11.1%, 15.2%, 19.4%, 16.6%, 23.6%, and 6.9%. Further, a strong correlation was observed between the abundance of β-lactamase-producing strains and IUS genes. Conclusions Based on our knowledge from this study, the association between β-lactamase production and IUS genes in A. baumannii plays an essential role in the emergence of drug-resistant strains.


Introduction
Acinetobacter baumannii (A. baumannii) has become an increasingly important human pathogen connected with infections acquired in hospitals [1]. It has become more challenging to treat A. baumannii infections in the hospitalize portions because of their resistance to significant groups of antimicrobial agents such as β-lactams [2]. Various resistance mechanisms make A. baumannii a successful pathogen of the 21 st century [3]. Extended-spectrum β-lactamases (ESBL) belong to the class A β-lactamases. Metallo β-lactamases (MBL), which have metal ions governing the active site, belong to the class B β-lactamases [4][5][6]. Class C β-lactamases are acinetobacter-derived cephalosporinases or the chromosomally encoded AmpC enzymes. Also, in class D β-lactamases, oxacillinases are involved in resistance to carbapenems. However, there is now a crucial need for new antibacterial strategies due to the spread of pathogen populations [7][8][9].
In A. baumannii, virulence factors and biofilm formation play a crucial role in disease progression in an infected host [10][11][12]. Another virulence factor studied in detail is A. baumannii to survive in an iron-limited environment, like the human host [13]. In response to low iron, A. baumannii produces the siderophore acinetobactin to acquire this essential micronutrient. A better understanding of the acinetobactin system could lead to new antimicrobial drugs to treat infections caused by multidrug-resistant (MDR) A. baumannii infections [10]. However, A. baumannii synthesizes siderophores which are relatively low-molecular-weight agents capable of converting polymeric ferric oxy-hydroxides to soluble iron chelates under low iron stress. e ability of bacteria to assimilate iron is related to invasiveness [3,14].
However, over the past decade, a need for new antimicrobial compounds has arisen with the advent of MDR bacteria, such as A. baumannii, plaguing our healthcare systems [13,15]. Since iron is essential for pathogenic bacteria to cause a successful infection, siderophore-mediated iron acquisition systems have been exploited as potential therapeutic targets [13]. One strategy utilizes the siderophore uptake machinery to deliver an antibiotic conjugated to the siderophore [2]. Nevertheless, in this study, to determine the relationship between β-lactamase enzymes and iron uptake pathways, we investigated the iron uptake system's role in the emergence of resistant strains.

Design of Study and Sample Collections.
A cross-sectional study was performed on patients admitted to Hamedan's Hospital, attached to Iran's Hamedan Medical School. e study was conducted between November 2018 and April 2020. Six hundred ninety clinical samples were collected, including wound swab, urine, blood, ascetic fluid, endotracheal fluid, bronchoalveolar lavage-inclusion criteria for patients presenting with signs of bacterial infection from bacterial culture examination. However, patients without bacterial infection symptoms, incomplete medical record data, or those who underwent nonstandard bacterial culture screening procedures were excluded from the study. After considering the inclusion and exclusion criteria, samples are used for culture and further biochemical investigations. Samples are inoculated in blood agar, MacConkey agar, and nutrient agar.

Detection of AmpC and MBL Producing Strains.
e AmpC and MBL producing A. baumannii were detected by the DDM [7].

Detection of Biofilm-Forming Strains.
Microtiter dish assay was used to identify the biofilm-forming A. baumannii [17].

Total DNA Extraction.
Genomic DNA was extracted using the method of Tahmasebi et al. [7]. By loading of 5 μl of the extracted DNA in 1% agarose gel, the extracted quality was checked.

Determination of Iron/Siderophore Mediate Gene.
According to Abdi et al. [3], the PCR method was used for the detection of the IUS gene. For partial gene, the PCR conditions used to amplify the iron/siderophore mediate gene were the same as described in Table 1. Moreover, PCR was performed using a C1001 Bio-Rad thermal cycler. Finally, 5 μl of amplified product is electrophoresed on 1.5% agarose gel at a constant of 80 V for 60 min [3].

Statistical Analysis.
In this study, GraphPad Prism software 8 (GraphPad Software, Inc., San Diego, CA) was used to analyze the data. Chi-square, two-way ANOVA, Tokay, and t-test were also used to examine the relationship between different variables. A p value of 0.05, 0.01, and 0.001 was considered as the level of significance.

Discussions
Acinetobacter's hospital infection has developed as a serious threat to the healthcare system due to the emergence of pan resistance from multiresistance. In recent years, A. baumannii has become a worldwide concern due to severe nosocomial infection.
In the current study, A. baumannii isolates obtained from several clinical samples were collected. Most samples were obtained from wound samples (31.9%) followed by urine (29.1%), blood (25.0%), and body fluids (13.8), which is concordance with study Lukovic et al. and Sheikh et al., where there are maximum isolates wound samples [18,19]. e current study revealed the lowest frequency of resistance to aztreonam (12.5%), followed by TMP/SMX (23%) and PIP/TAZ (25%).
is finding was also reported by Armin et al. and Kamali et al. [20,21]. In line with Jasemi et al.'s [22] study, 27.7% and 36.1% of isolates were considered XDR and MDR, respectively. However, these findings were in contrast to the data reported from India, Sweden, and Italy [4,23,24]. Also, 27.7% and 36.1% of A. baumannii produced AmpC and MBL enzymes, respectively. A similar pattern of results was obtained in El-  Figure 3: Correlation between the iron/siderophore mediate gene, biofilm formation, separated clinical isolates, and antibiotic resistance in A. baumannii. (a) Correlation between antibiotic resistance, biofilm formation, and iron/siderophore mediate gene. (b) Correlation between β-lactamase enzymes and iron/siderophore mediate gene. (c) Correlation between clinical sample type and iron/siderophore mediate gene. Each data set was analyzed using the Student's t-test, Tokay, and the two-way ANOVA and was presented as mean + SEM. * p value <0.05; * * p value <0.01; * * * p value <0.001; * * * * p value <0.0001. ns: nonsense.
International Journal of Microbiology Bakyet al. and Kaur and Singh. ey showed that about 35% of A. baumannii isolates produced AmpC and MBL enzymes [8,25]. However, these findings contrast with the previous studies in India and Pakistan [26,27]. Also, by the time A. baumannii isolates acquire resistance to β-lactams and carbapenems, they often acquire resistance to several other antimicrobial agents as well [26]. According to the present study, the frequencies of basD, bauA, pld, paaE, feoB, hemO, tonB, and entA genes in A. baumannii clinical isolates were 12.5%, 15.2%, 11.1%, 15.2%, 16.6%, 23.6%, 6.9%, and 19.4%, respectively. However, this frequency has not previously been described. e levels observed in this investigation are far below those observed by Abdi et al. [3]. ey reported that frequencies of tonB, barA, feoB, entA, and hemO genes were 85%, 97%, 99%, 99%, and 95%, respectively. A study conducted by Liu et al. [2] also showed a high prevalence of iron/siderophore genes in A. baumannii isolates. Unfortunately, few studies have surveyed the abundance of iron/siderophore mediate genes.
A significant relationship was observed between antibiotic resistance and IUS genes' distribution based on our study (p < 0.05). However, there are very few nationwide studies conducted on IUS genes associated with antibiotic resistance and biofilm formation in Iran. Furthermore, we found that AmpC/MBL producing among A. baumannii isolates is significantly correlated with increased biofilm formation (p < 0.001). A correlation between IUS and antibiotic resistance patterns was also reported by Zeighami et al. Moreover, these authors reported a correlation between IUS and upregulation of the β-lactamase gene encoding the AmpC and MBL enzymes. ey suggested that IUS played an essential role in the prevalence of antibiotic resistance in clinical isolates of A. baumannii [28]. Moreover, following the present results, Kröger et al. and Mohajeri et al. have demonstrated that iron uptake through siderophores' production is another factor of virulence in this organism. However, this finding broadly supports the work of other studies in this area linking iron/siderophore mediate with drug resistance [13,28,29].
A. baumannii isolates by several mechanisms of resistance can also deferentially express various virulence factors. ese factors include siderophore production, biofilm formation, and hemolysis on blood agar [13,30]. However, the association between virulence and antimicrobial resistance seems to be a highly complex one that is still unclear. Notably, the effect of harboring β-lactamase on virulence in A. baumannii is not well defined.
Finally, an association between IUS and biofilm formation on the one hand and biofilm formation and antibiotic resistance on the other was determined. e relationship between β-lactamase production and biofilm formation has been previously reported among MDR A. baumannii isolates [12,28,31]. Moreover, the association between motility and siderophore production is not surprising since the former factor was associated with biofilm production. Simultaneously, the latter allows for iron acquisition that is crucial for biofilm formation [14]. ese associations reveal a highly complex interplay between the different virulence determinants in A. baumannii, especially multifactorial. erefore, considering this relationship, drug-resistant strains can be regarded as more prone to invasion. On the other hand, A. baumannii reduces its sensitivity to antibiotics by using iron/siderophore pathway regulatory systems.
In the present study, IUS pathways in antibiotic resistance in clinical isolates of A. baumannii were identified. We demonstrated that iron/siderophore uptake plays an essential role in the prevalence of AmpC/MBL producing strains among A. baumannii isolated from clinical specimens. Besides, the presence of feoB, entA, and hemO genes was associated with increased virulence and antibiotic resistance compared to pld, and tonB was positively associated with AmpC/MBLand siderophore production. Nevertheless, our results confirmed that IUS pathways significantly affect the forming extracellular layers in A. baumannii.
us, suppression of feoB, entA, and hemO genes is one of the best ways to control virulent strains. It also inhibits the iron/ siderophore uptake pathway increased biofilm-forming strains' sensitivity to the β-lactams antibiotics. Further, these associations could help better understand the complex interactions between antimicrobial resistance and virulence.
Data Availability e data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare no competing interest.