Identification of Acinetobacter baumannii and Determination of MDR and XDR Strains

The current study focuses on the bacterium Acinetobacter baumannii due to its importance as a nosocomial infections source in addition to its increased resistance against antibiotics. Different clinical and hospital environment samples were collected, and cultured on A. baumannii selective media: Leed Acinetobacter agar and Herellea agar. A. baumannii have been identified by traditional methods, followed by confirmation using molecular identification to detect blaoxa-51 like gene which is considered a diagnostic gene since it is present in genome of all A. baumannii strains. The result was, nineteen bacterial isolates of A.baumannii were obtained, from twenty-seven suspected isolates, detection of local isolates belonging to MDR or XDR group. Results demonstrated that all local isolates are MDR and 16 isolates (84.2 %) are XDR.


Introduction:
Acinetobacter baumannii (A. baumannii) is a gram negative bacteria, sometimes diagnosed as gram positive due to its resistance to alcohol when stained with gram stain, strict aerobic, non-motile but has some sort of twitching motility by polar fimbriae.
Oxidase negative, catalase positive, indole negative, positive for citrate with C/G content 47-39%, non-lactose fermenter, the optimum temperature for their growth is 33-35 o C, many strains are nitrate negative and do not form spores They are easy to grow on the non-fastidious culture media (1,2) A.baumannii bacteria have been of increasing importance for several reasons: they cause hospital infections and are responsible for 2-10% of all hospital-related infections of gram-negative bacteria, like septicemia, meningitis, UTI and others. They are an opportunistic pathogen, as well as for the acquisition of multi drug-resistant (MDR) and extensive drug-resistant (XDR) (3). This bacterium is resistant to many antibiotics, including aminoglycosides, tetracyclines, cephalosporins, ampicillins, cefotaximes, chloramphenicol, gentamycin, tobramycines, quinolones and marcolides (4,5).
The main cause of A.baumannii infections is due to: speed in transformation, rapid development in resisting a wide range of antibiotics such as efflux pumps system and horizontal gene transfer, and prevalence in the dry environment of hospitals for long periods of time (6).
This study aims to investigate the prevalence of A.baumannii isolates by implementing special selective media, identification by conventional methods followed by detection of the bla oxa-51 gene (diagnostic gene for A.baumannii because it is present in the genome of all its isolates ), and that delimiting MDR and XDR A.baumannii isolates.
including samples from intensive care unit, patient beds, surgical instruments and appliances, emergency lobby and baby incubators.
The specimens were transferred to the laboratory (by transport media) and cultured on selective A. baumannii media (Leed Acinetobacter medium (LAM) and Herellea agar medium) prepared according to (7,8). As well as blood agar and MacConkey agar then incubated at 37°C for 24 hours under aerobic conditions.

Identification: Cultural characteristics and microscopy:
The cells were examined microscopically to observe the staining reaction and cells arrangement. primers of the USA Alpha Company, the primer has a molecular size of 353 base pairs and consists of sequences: bla OXA51 F:5 TAATGCTTTGATCGGCCTTG 3̀ bla OXA51 R:3̀ TGGATTGCACTTCATCTTGG 5̀ PCR mixture was prepared as the total size 25 microliters, 4 µL DNA (50 µL), primers (10 picomol), 6.5 µL Premix (2x) and 12.5µL Deionized water. The previous prepared mixture was placed in thermal cycler, and the program was run as suggested by (11) :94 • C for 5 min., 94 • C for 30 sec., 55 • C for 30 sec., 72 • C for 90 sec (30 Cycles), 72 • C for 7min.

Results and Discussion: Morphological result:
After taking the samples to the laboratory, they were cultured on A. baumannii selective media, the colonies of this bacteria on Leed Acinetobacter agar(LAM) showed a convex circular shape with smooth transluscent opaque with smooth edges of 1-2 mm diameter and pink color with pink to red background after 24 hours of incubation at 37°C (Fig. 1). The transformation of the color of the medium to red is due to the high alkali produced in the medium by the release of ammonium ions from complex nitrogen compounds in the medium. A.baumannii produces alkaline compounds when sucrose and fructose are consumed in the medium making the color of the medium red (9,12).
A.baumannii colonies on the Herellea agar ( Fig. 2) were round with smooth margins of 2-3 mm in the form of pale lavender flower, this is similar to what is described by (7,8).
While colonies on MacConkey agar were pale, round and with a diameter of 2-3 mm because it is non-lactose fermenter and turned pink after 48 hours of incubation as in Fig. (3), and this description is consistent with (1). On blood agar, colonies were convex, gray or white color. It does 728 not lyse blood because it does not producing the enzyme hemolysin as shown in the Fig. (4).

Biochemical tests
The results of biochemical tests for identification of A. baumannii are shown in Table  (1).

Diagnosis with API-20E:
The code was entered into the analytical profile index to find out the full scientific name for bacterial isolation, as in Fig.5.  bla oxa-51 like gene is specific for A.baumannii species and is present on the chromosome (13). It is a diagnostic gene of A. baumannii but also has a significant role in resistance to antibiotics, because it is related to the resistance of carbapenem through the production of enzymes Carbapenemases, the secondary group of these enzymes is Class D carbapenem (oxa51), also called Oxacillinase naturally located on the chromosome of this bacteria (14,15).
The percentage of A. baumannii isolated in our study was 8.1 %(from 19 isolate of A.baumannii obtained from 233 sample), the isolates have been distributed between clinical (15) isolates and hospital environment (4) isolates (Table 1). Most isolates were from wound samples followed by respiratory tract infections, and none A. baumannii isolates were obtained from urine samples. The number of samples and geographical distribution of it may be the cause for different the ratio with other studies. The results of our study agreed with the study of (16) in Baghdad as the percentage of isolation of A.baumannii was 8.2% compared in the present study. Also, Al-Dulaimi et al. (17) in Babel obtained 7% percentage of A. baumannii isolates from wound infections. Isolation percentage in the current work was higher than the rate of isolation of Al Sehlawi et al. (18) in the city of Najaf in 2014, as it was 1.9% of the total samples of wounds, but less than the isolation rate of (19) in Iran 24% isolation was obtained from respiratory infection.

Presence of MDR and XDR for isolates A.baumannii
For the purpose of determining which of our local isolates of A.baumannii belong to the MDR or XDR group, the sensitivity of the 19 bacterial isolates was first tested against 15 commonly used antibiotics. The diameter of the inhibition area was determined and compared with the tables of CLSI to determine resistance, sensitivity and moderate sensitivity (20). Table 3 shows the ratio of sensitive isolates, resistance and average sensitivity in antibiotic susceptibility test. The results in Table (3) showed a high resistance of A.baumannii isolates to antibiotics, all 19 isolates were resistance 8 antibiotics(100%), while 17/19 isolates were resistance 3 antibiotics with the rate (89.4%) . A.baumannii bacteria have become important and a source of study by doctors and scientists because of the rapid spread of antibiotic resistance and the slow development of new antibiotics (21).
The results of our study were consistent with the study of AL-Kadmy et al. (22), It showed 100% resistance of A.baumannii for Ciprofloxacine and Trimethoprime, the resistance to Cefetriaxone, Tobramycin, Tetracycline and B-lactams was more than 90% .While the resistance to isolates of the antibiotic Meropenem is 86% .This coincides with the results of the current study, which gave a100% resistance to this antibiotic. Also, the results of our study were consistent with the results of the Azizi et al. (23) in terms of resistance to Ciprofloxacine and Gentamicin as it was 100% each of them.
When isolate was resistant to 3 classes of different antibiotics classified as MDR, the isolate was resistance of most antibiotics class except one or two groups classified as XDR The results of our study showed 100% MDR-Ab, and that MDR was known to be at least resistant to three classes of antibiotics, such as penicillins, cephalosporins, fluroquinolones, and aminoglycosides.
Sixteen isolates (84.2%) of XDR-Ab were obtained, XDR is the isolating resistance of almost all antibiotic classes except one or two groups.
Our results matched the results of (24), with the MDR rate being 100%, and the XDR ratio of our isolates was higher ( 62.8% ).
The MDR ratio was in agreement with that of (23).
The high resistance of isolates obtained in the present work may be due to several mechanisms: Aminoglycosides-modifying enzyme, Production of B-lactamases enzymes, Reduction in the expression of (altered) outer membrane proteins, Mutations in Topoisomerases, Work on increasing the organization of Efflux-pumps (25).
The results of the molecular diagnosis of bla oxa-51 gene confirmed the results of our study on MDR where all isolates had this gene, and that the results of this study confirmed that XDR-Ab is the largest antimicrobial antibiotic that must be treated and controlled in hospitals and health centers (6)

Conclusion:
A.baumannii can grow on simple culture media prepared in the laboratory (nonfastidious). It was called the term "MDR" and "XDR" because of its resistance to many antibiotics, as well as its possession of antibiotic-resistant genes such as a gene bla OXA 51-like which is a fundamental gene for the diagnosis of this type of bacteria.

Authors' declaration:
-Conflicts of Interest: None. -Ethical Clearance: The project was approved by the local ethical committee in University of Mosul.