Molecular Characterization of Multiple Antibiotic- Resistant Acinetobacter baumannii Isolated from Egyptian Patients

Acinetobacter baumannii is an opportunistic microorganism commonly found in intensive care units (ICUs), and it is responsible for a broad span of hospital-acquired infections. Persistence of nosocomial infection caused by multidrug-resistant (MDR) A. baumannii is an alarming health care issue in Egypt, and at present, colistin remains the treatment of choice for the management of MDR A. baumannii infections. A. baumannii possesses great capacity to develop and acquire resistance to a broad range of antibiotics. The acquisition and dissemination of antibiotic-resistant determinants in A. baumannii strains are mediated by integrons, especially class I integrons. This study focuses on the characterization of some genetic mechanisms underlying the multidrug-resistant phenotypes of A. baumannii isolates in Egypt. Forty-eight A. baumannii specimens were isolated from different hospitalized patients; least resistance was observed against amikacin and tigecycline, with 60% and 58.5% of the isolates resistant, respectively, whereas 62.5% of the isolates were resistant to imipenem and meropenem. The highest sensitivity was found for colistin. Genetic analysis revealed that blaoxa-51 was detected in all isolates, the blaoxa-23-like gene was detected in 80% of the isolates, and blaoxa-24 and blaoxs-58 were not detected in any isolate. Finally, PCR analysis revealed that 6.6% of isolates carried the class I integron gene.


InTRoDUCTIon
A c i n eto b a c te r b a u m a n n i i i s a n opportunistic microorganism commonly found in intensive care units (ICUs). It is responsible for a broad range of infections such as urinary tract infections, surgical wound infections, ventilator-associated pneumonia, meningitis, bacteremia, and other life-threatening infections. The primary factor leading to the wide spread of this nosocomial microorganism is its capacity for possessing a broad spectrum of antibiotic resistance genes. Selective pressure due to environmental stress causes the prevalence of these antibiotic-resistant clones 1 . Its broad assortment of antimicrobial resistance genes makes infections difficult to treat, thereby causing financial burdens that ultimately create problems in infection management in hospitals. The quickly increasing prevalence of multi-drug resistant A. baumannii isolates is becoming a significant concern in global public health. The spread of resistant genes in hospitals and in society is caused by horizontal gene transfer 2 and movable segments such as plasmids, transposons, and integrons are the primary genetic elements responsible for the spread and prevalence of these resistance genes 3 . Over the past decades, despite the discovery of new drugs and treatment choices, A. baumannii strains have been shown to possess a capability to rapidly evolve multidrug resistance (MDR). This rapid increase in MDR is not only caused by the inherent resistance genes carried by these strains, but also by their extraordinary capacity to obtain resistance elements from other microbes. The roles of efflux pumps, class B β-lactamase (metallo-β-lactamase), chromosomal class C β-lactamase, AmpC, class D β-lactamase (OXA-type carbapenemase), integrons, and associated insertion sequence elements in MDR occurrence have recently been well documented 3 . The acquisition and spread of antimicrobial resistance determinants in multidrug-resistant A. baumannii strains are frequently mediated by integrons, especially class I integrons. Integrons are a key element in MDR spread, particularly in gram-negative pathogens 4 . These are usually immobile, but they can be transported through mobile genetic elements such as plasmids and transposons. Integrons have the ability to merge into the microbial genome, and they possess numerous gene cassettes that confer antibiotic resistance 5 . Integrons can be categorized into three segments based on sequence conservation: the 5'-conserved segment, the variable region, and the 3'-conserved segment. The 5'-conserved segment contains a promoter, P C , and an intI gene encoding integrase; the variable region usually incorporates various gene cassettes, and the 3'-conserved segment comprises sequences acquired from transposons such as qacEΔ, sul1, and orf5, which are found in class I integrons 6 . Integrons carry divergent gene cassettes that are rearranged under antibiotic selective pressure 7 . In A. baumannii, these gene cassettes frequently comprise efflux pump genes, β-lactam resistance genes, and aminoglycoside resistance genes. Up to now, a few categories of integrons have been recognized based on the succession of the integrase gene. Among these categories, class I integrons are the most common class type, and these are fundamental in the development and spread of resistance genes, followed by class II. Reports on the different classes of integrons are scarce 4,8 , though based on literature, class 1 integrons and the pool of associated gene cassettes are the significant causes of A. baumannii MDR and could thus become a valuable instrument for studying molecular epidemiology in possible crossinfection cases, particularly in critical wards of hospitals such as ICUs 2 .

Bacterial isolates
Our study was conducted from 2016 to 2019. Forty-eight non-repeated A. baumannii isolates were collected from various clinical samples of hospitalized patients in El-Fayoum General Hospital. Isolates were identified using Gram staining, followed by specific biochemical reactions to identify Acinetobacter up to the genus level 9 . The isolates were then confirmed using the VITEK 2 system, following the manufacturers' instructions 10 . Finally, isolates were confirmed by examination of the bla oxa_51_like gene, which is intrinsic to A. baumannii 11 .

Antimicrobial sensitivity testing
In vitro sensitivity tests were carried out using a panel of 11 antibiotics for all isolates using the disk diffusion method, in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) 12 . The following antibiotic disks were used: amoxicillin/ clavulanate (AMC), piperacillin/tazobactam (TPZ), cefoxitin (FOX), cefotaxime (CTX), cefepime (FEP), imipenem (IPM), aztreonam (ATM), ciprofloxacin (CIP), tigecycline (TGC), amikacin (AK), and colistin (COL). The standard reference strain Escherichia coli ATCC 25922 was used as a quality control strain in every test run.

Determination of the minimum inhibitory concentration (MIC)
MIC was tested for each isolate using the broth dilution method, as described by Irith Wiegand 13 . Bacteria were cultured in Muller-Hinton broth with a serial dilution of antibiotics. Growth (turbidity) was then determined after incubation for a specific duration (16-20 h), and the MIC value was then determined.

Extraction of genomic DnA
A. baumannii isolates were refreshed by culturing in Luria-Bertani (LB) broth and incubating overnight at 37°C. Fresh culture was then used for DNA extraction by boiling 1 mL of each isolate in LB suspension in an Eppendorf tube at 100°C for 5 min in a water bath to lyse the bacterial cell wall and liberate the DNA into the medium. The tubes were then centrifuged at maximum speed for 5 min, and the supernatant was then utilized for PCR 14 .

Detection of integron genes in A. baumannii isolates
For mining integrons, the following primers were used: intI1, intI2, and intI3 (Table  1) 15 . PCR reactions were performed using 5 μL of DNA, 0.2 mM of each type of deoxynucleoside triphosphate (dNTP), and 2 μL of 10× PCR buffer. One unit of Taq polymerase, 1.5 mM MgCl 2 , and 1.25 μM of each primer were then added to the reaction. PCR conditions used were as follows: 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, 30 s of extension at 72°C, with a final extension at 72°C for 10 min 16 .

Detection of carbapenem resistance genes
PCR was carried out in a reaction mixture containing 1× PCR buffer with 1.5 mM MgCl 2 , 0.2 mM dNTP, 1.5 U Taq polymerase, 3 μL DNA, and 50 pmol of forward and reverse primers per reaction. Primer sequences used to amplify genes encoding bla oxa-23 like, bla oxa-24 like, bla oxa-51 , and bla oxa-58 like genes are summarized in Table  1. The PCR conditions used to amplify OXAtype carbapenemases were as follows: initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 25 s, 52°C for 40 s, and 72°C for 50 s, with a final elongation step at 72 °C for 6 min 17 .

Bacterial isolates
All isolates showed gram-negative coccobacilli arranged in diploids. The bla oxa-51like gene was found in each isolate as a PCR product of 353 base pairs (bp).
Antimicrobial sensitivity of A. baumannii isolates using the disk diffusion method with E. coli ATCC 25922 as the control strain showed resistance patterns as follows: ( The MIC values for carbapenems were above 32 μg/mL for (30) Table 2.

Integron detection in A. baumannii isolates
Two isolates were found to possess the class I integron gene, as shown in Fig. 1. None of the class II or class III integrons were detected.

Detection of carbapenem resistance genes
All carbapenem-resistant A. baumannii isolates were subjected to PCR screening for the presence of CHDLs. The bla oxa-51-like gene was detected in all isolates, bla oxa-23-like gene was detected in 24 isolates, and the bla oxa-24-like and bla oxa-58-like genes were not detected (Fig 2).

DISCUSSIon
A total of 48 Acinetobacter clinical samples were tested for antimicrobial susceptibility, and all were found to be greater than 58% resistant to all the selected antibiotics. Ninety percent of the samples were resistant to amoxicillinclavulanate, 96% resistant to second-generation cephalosporin, 98% resistant to third-generation cephalosporin, 88% resistant to fourth-generation cephalosporin, 62.5% resistant to carbapenems and monobactam, 64.5% resistant to quinolone,  and 60.4% to resistant amikacin. In contrast, all isolates showed 0% resistance to colistin. These data indicate the extreme drug-resistant risk of nosocomial infections, consistent with data from the Middle East, published by Al-Sweih NA 18 and with previous results obtained from Egypt 19 . In our study, the respiratory tract was the primary site of infection, which is similar to data published by Joly-Guillou ML 20 . Moreover, we found that colistin is the only treatment option left for managing A. baumannii infections.
MIC values for carbapenems were above 32 μg/mL for 62.5% of isolates, and above 128 μg/ mL, 256 μg/mL and 128 μg/mL for 91.6% of secondgeneration, 97.9% for third-generation, and 87.5% for fourth-generation cephalosporins. Amoxicillin/ clavulanate and piperacillin/tazobactam showed MIC values above 256 μg/mL for 75% and 70.8% of isolates, respectively. On the other hand, monobactam revealed values above 256 μg/ mL for 64.5% of isolates and above 256 μg/mL for amikacin and ciprofloxacin; these data are consistent with data from Egypt published by Fouad et al. 21 Based on the obtained sensitivity and MIC data, all carbapenem-resistant A. baumannii (CRAB) isolates were subjected to further investigation.
The most common carbapenemases in A. baumannii are the carbapenem hydrolyzing class D β-lactamases (CHDLs) and metallo-β-lactamase (MBL), while Class A carbapenemases were found at a lesser extent 22 . CHDLs can be classified into four main subgroups: the intrinsic bla oxa-51-like  The bla oxa-51like gene is intrinsic to A. baumannii 17,23 , and its discovery in all isolates of the present study confirmed their identity, as In this study, multiplex PCR showed that 24/30 (80%) of CRAB isolates harbored bla oxa-23-like genes, whereas bla oxa-24/40-like and bla oxa-58-like genes were not detected, consistent with numerous studies that reported bla oxa-23-like as the most prevalent carbapenemase among carbapenemresistant A. baumannii [26][27][28] . In Egypt, Fouad et al. confirmed the importance of the bla oxa23-like gene among A. baumannii isolates 21 .
The class I integron has been documented in several studies of multi-drug resistant A. baumannii worldwide 29 . In this study, 6.6% of CRAB isolates were positive for class I integrons, while class II and class III integrons were not detected; this is consistent with a study performed in Turkey, in which 6.4% CRAB isolates were positive for class I integrons 30 . On the other hand, our results are not consistent with that of Mehdi et al., who reported a significant prevalence of class I and class II integrons 31 .

CONClusiON
Acinetobacter baumannii poses a serious threat in Egyptian hospitals, and our current findings suggest a critical healthcare problem that can lead to the loss of effectivity of future therapeutic options. As such, stricter infection control measures are required to decrease intrahospital and interhospital spread of multidrug resistant A. baumannii.