Occurrence of Metallo-β-lactamase Genes among Acinetobacter baumannii Isolated from Different Clinical samples

Acinetobacter baumannii became a serious endemic and widespread pathogen responsible for causing nosocomial infections due to restricted treatment options. This study was conducted to evaluate the role of both antibiotic resistance and genetic pattern in infection caused by MBLs resistance A. baumannii isolated in Baghdad hospitals. Finally, developing selective media in order to detect multidrug resistance A. baumannii isolates according to colony shape appearance changing. Collected 124 isolates from various clinical and environmental sources. Biofilm Formation was detected, Antibiotic sensitive for 21 antibiotic discs were determined, MDR index calculated, PCR was performed to investigate MBLs genes bla (IMP2, KPC, DIM, SPM, VIM, GIM, AIM, BIC, SIM, NDM, Pre-NDM-A, GES, Imp-1, and VIM). Increasing resistant prevalence of A. baumannii appear significantly in higher rates to seven antibiotics group and MDR index ranged between 0.29 to 1.0 from 6-21 antibiotics. It was clear in Biofilm formation evaluated 75.8% strong biofilm after 72 hours, indicated that there is a significant negative correlation between biofilm formation capacity and the inherent ability of bacteria to show multidrug resist (P< 0.001). Our findings indicated that non-MDR isolates tended to form more robust biofilm formation. Metallo β-Lactamase detection phenotype showed 113/124 isolates able to produced ESBL, a result confirmed by PCR assay to detect the resistance of MBL-genes, appeared that a high percentage of blaIMP 79% which left clinicians with limited treatment options. An increasing number of hospital MDR A. baumannii has been reported. Furthermore, the study will focus on the evolution and the emergence of multidrug resistance A. baumannii in Iraq. The common MBL gene is the blaIMP.


iNtRODUCtiON
Acinetobacter baumannii is one of the most important opportunistic pathogens that cause outbreaks in hospitals, health care and also associated with complications in hospitalized patients 1 .
Unstable genetic element, tolerate harsh environments, and Metallo ‫-ג‬lactamases (MBLs) enzyme have led to the development of MDR resistance and epidemic A. baumannii 2,4 .
Different mechanisms of resistance and resistant to all commercial antibiotic caused alarming problems in a limited choice of antibiotic for the treatment of MDR. A. baumannii isolates 3 .
Increasing of MDR hospital isolates A. baumannii isolate has been stated from many countries around the world. Plus, inter-hospital environment dispersal of multidrug-resistant A. baumannii has been observed 5 . The ability of A. baumannii to rapid genetic change owing to their ability to acquire genes horizontally and vertically 6 .
This study came to highlight the current patterns of resistance to antibiotics as well as the prevailing genetic patterns of resistance in A. baumannii in order to achieve effective treatment in Baghdad hospitals for these bacteria with multiple resistance to antibiotics, which represents a threat to public health.

Sample collection
The study was carried out from December 2017 through March 2018 and involved 6 hospitals: Children's Educational Hospital, Baghdad Teaching Hospital, Al-Hawraq Hospital, Educational Laboratories (Medical City), Imam Ali Hospital and Al-Kindi Hospital. After the patients were examined by the physician, samples were collected from various sources: wounds, burns, ulcers, spinal cord fluid-blood, blood, body fluid and swabs of the hospital environments and tested using conventional microbiological methods.

Laboratory Identification of Isolates Morphological Examination:
The samples were diagnosed including Gram staining, traditional culture and using the selective media such as the chromo agar Acinetobacter medium and use modified Leeds agar base media by replacing fructose and sucrose and Manitol by xylose and glucose and add crystal violet dry powder dispense from selective media. Furthermore, the results of the identification of A. baumannii were confirmed by the API 20E system according to Forbes 7 .

Temple preparation for PCR
Template DNA was prepared by the boiling method as described by 8 .

Molecular detection of A. baumannii
In order to confirm the detection, isolates were subjected to PCR against the 16s rRNA, oxa51 and RecA, PCR mixture and amplification were done as explained by 9,10,11 respectively.

Modified Hodge test (MHT)
Performed according to Bonnin 12 , for detection of carbapenemase production, Metallo b-Lactamase and ESBLs production among isolates by using modified indirect three-dimensional methods according 3 .

Biofilm Formation
Congo red agar (CRA) method (qualitative Biofilm production assay): A simple qualitative assay for detection of biofilm was described by Mathur 13 .With modification as follows: (40) g\l blood agar base, (0.8) mg Congo red,(5)g\l d-glucose, (5) g/l xylose. These components were dissolved in 1L of distilled water and autoclaved. The agar then was dispensed into sterile Petri dishes and kept at 4°C until being used. Following inoculation, the agar plates were incubated at 37°C for 24-48 h. The appearance of black colonies with a dry crystalline consistency could be considered as strong evidence for the ability to form a biofilm. Each experiment was conducted in three repeats Tissue culture plate method (quantitative biofilm production assay) Generally considered to be the goldstandard technique for biofilm detection, Adopted the prescribed route by Lotfi 14 .

Motility Assay
Motility Assay Performed as per 15 . PCR amplification of Metallo β -lactam genes: PCR method was used for screening of the Metallo β-lactam genes: bla (IMP2, KPC, DIM, SPM, VIM, GIM, AIM, BIC, SIM, NDM, Pre-NDM-A, GES, Imp-1, and VIM). The primers and PCR programs used in this study were as previously described by (16; 17; 18 and 5 ). PCR product bands were analyzed after electrophoresis in a 1% agarose gel at 95 V for 45 min in 1X TBE containing Ethidium Bromide under UV radiation 8 .

Statistical analysis
O n e -w ay A N O VA s w a s a p p l i e d exploitation SPSS 21 so as to match variations among Microtiter plate below standard and changed conditions. The experiments were performed in triplicate. P values of ≤ 0.05 were thought of as significant 31 .

Antimicrobial susceptibility detection
The overall susceptibility patterns of A. baumannii isolates from various clinical sources is displayed in Table 1. Isolates showed high XDR 75%, MDR 23.4%, and PDR 1.6%, The MAR index for experimental isolates (6-21 from 21 antibiotics) ranged between 0.29 to 1.0. β-Lactamase detection M eta l l o β -L a c ta m a s e d ete c t i o n phenotype showed (n=113)91.1% isolates able to produced while only (n=1)0.8% overall the isolates produce ESBL.

Correlation between biofilm formation and multidrug-resistant isolates
The simple logistic regression test analysis indicated that there is a significant negative correlation between biofilm formation capacity and the inherent ability of bacteria to show multidrug -resistance index (P < 0.001). Our findings indicated that non-MDR isolates tended to form more robust biofilm formation Fig. (3). Among 124 strong biofilm producers, 94% (n = 91) were non-MDR and 6.2%% (n = 6) were MDR isolates. All of the negative biofilm isolates were MDR 100% (n=3) and weak biofilm producers were 50%MDR index isolates and 50% non-MDR.

Red color means constant
Journal of Pure and Applied Microbiology showed an average migration area of between 5 and 25 mm.

Association between bla genes, antibiotic sensitivity, and biofilm formation
The result can be divided to 4 group description as shown in Table 2, Hence, the most prevalent group was D include strong bioflim production with highly MDR index and different patterns of gene resistant.
Allocation of specimens of A. baumannii may diverge with each hospital as each hospital aperient has a different environment associated with it. More than 91.94% of the A. baumannii isolates were obtained from CSF culture, Blood culture, burn swab, postoperative wound swab, sputum, urine, and body fluid Similar results had been obtained in different studies reported in Iraq by Hussein (19;5) and (15). As well in different countries such as Doughari 20, 21,22 .
This consideration reveals that a total 76/124 A. baumannii were isolated, d from systemic infection CSF and blood culture, This may be due to the fact that bacteria are opportunistic pathogens capable of penetrating the defenses of the body, although ability to adhere to the instruments or hands of workers, which makes it easier to transmitted between patients who are in hospitals, from above indicated horizontal transition of bacteria from the skin or infected organs to the blood causing Bacteremia.
The use of selective cultures in the diagnosis of A. baumannii in principle is one of the quickest methods and gives a good percentage of diagnosis. This study proved the modified media in the accurate diagnosis of A. baumannii compared to the routine culture media. On the other hand, the study clarified that the diagnosis of genus and species together by using the PCR reaction is quick, It is clear that the emergence of resistant A. baumannii isolates is increasing worldwide. In this research, the resistance of A. baumannii isolates to 15 out of 21tested antibiotics was above 80%.
The frequency of MβLs among isolates of A. baumannii in this study was 91.1%. In M ‫ג‬L-producing A. baumannii were multidrug resistant, the lowest resistance was observed against Tetracyclin and doxycycline which can be used as a treatment option. A significant increase in resistance to carbapenem antibiotics was observed, In this regard 23 pointed out that the most important reasons for the emergence of resistance to carbapenem antibiotics are including the production of enzymes Carbapenemases belonging to β-lactamase enzymes, and loss of protein in the outer membrane has been associated with anti-microbial resistance to Imipenem and Meropenem. It is worth mentioning that the resistance of Meropenem was higher than Imipenem. The high rate of resistance to most cephalosporins antibiotics tested indicates these bacteria possess multiple resistance mechanisms. In addition to their production enzymes β-lactamase and extend β-lactamase and have the ability to alter outer membrane proteins and efflux pump system that acts on efflux the antibiotic extracellular extrusion 24 . The resistance of A. baumannii to the quinolones and aminoglycoside was increased even to an active antibiotic such as Amikacin.
The study demonstrated the increase of antibiotic resistance to most classes of antibiotics, especially the antibiotics of choice for the treatment of A. baumannii infections include the fluoroquinolones, aminoglycosides, and carbapenems.
The MAR index values for all isolates showed high MDR, ranged between 6-21 from 21 antibiotics. The MAR index for experimental isolates ranged higher than 0.29 reach to 1.0. Suggesting the source of these isolates is from highly contaminated environments where it is excessive and randomly use of antimicrobial agents. Development of MAR pathogenic isolates of A. baumannii determined potential nosocomial infection in the hospital environment. Most isolates from CSF and respiratory system had MAR indices over (0.43), and over (0.76) show in UTI infection while the lowest MDR index (0.29) in body fluid confirming that there was a high antibiotic use and high selective pressure in these environments MDR strains become established in the hospital environment, these can persist for months.

Detection of biofilm formation Microtiter plate assay
A. baumannii isolates were classified into strong, moderate, weak, and none biofilm producers, respectively at 550nm.Gentile 25 . demonstrated that there was a high rate of biofilm production between 60% of highly resistant A. baumannii isolates, our results agreed with Iraqi study 15 .

Twitching motility pattern
It was believed in A. baumannii strains that display a motility phenotype depending on autoinducer molecules related to phenomenon quorum sensing.McQueary 26 . Further revealed that the production of lipopolysaccharide (LPS) Pili has an important role in twitching motility and adhesion (inert or living surface) and contributes indicating biofilm formation. The results indicated that when the resistance to antimicrobial agent rate is relatively high, there is an increase in productivity of adhesion factors gradually, on the contrary of Ali and Khudhair 27 study.

Genotype detection of the studied genes for MBLs
In our study, blaBICwere identified only in (21.8%) of A. baumannii isolates by PCR. In the other hand, the bla KPC gene was identified among 16 /124 isolate (12.9%) of A. baumannii. Also, the bla GES gene was detected among 32/124 isolate (25.8%) A. baumannii. Class A β-lactamases in A. baumannii may be considered a major problem in comparison to other carbapenemases and was usually not-carbapenem specific and often hydrolyze most compounds of the lower betalactam classes and take part in the high-level resistance to carbapenems 28 .
The high frequency of Class A carbapenemases genes incidence may be because they are plasmid mediated so bacteria can gain this plasmid from another gram-negative bacteria presented in hospital environmental.