Genotyping of fusA Gene from Clinical Isolates Acinetobacter baumannii in Baghdad

This study aims at detecting the differences in genotyping of coding region fusA gene in clinical isolates of Acinetobacter baumannii from Baghdad, Iraq. Collected two hundred clinical samples (50 samples from urine, 50 samples from wound, 50 samples from sputum and 50 samples from otitis infections). Laboratory diagnosis for bacterial isolates carried out by some biochemical tests and confirmed by using VITEK2 compact system. The results appeared that twenty isolates of Acinetobacter baumannii in all these samples. Genotyping study was performed of coding region fusA gene of the extracted genome of all bacterial isolates and used specific primers in achieved amplification process of this target gene. DNA sequencing of this gene and alignment of sequencing in NCBI was achieved and drew phylogenetic tree by using Geneious 9 software among locally isolates alone and then among locally isolates and high identity global isolates in GenBank. The results in phylogenetic tree of fusA gene in locally isolates showed 4 groups of isolates included more than one source of isolation. The results in phylogenetic tree of the locally and global isolates showed that are four different groups and each group included some locally isolates and global isolates except group A (AE_22, AE_26) and group E (AE_35, AE_32, AE_33) that not identity with global isolates. The nucleotides sequence of fusA gene from localized isolate (AE_35) was registered in national GenBank under accession number (LOCUS KY818057) and protein ID "ARV90995.1.


Introduction:
The bacterium A. baumannii is an opportunistic pathogen (Gram-negative bacilli bacteria) which causing world wide nosocomial infections included different of hospital acquired infection (UTI, endocarditis, surgical-site infections, meningitis, septicemia, and ventilator-associated pneumonia among patients in ICU) (1,2,3).These bacteria develop resistance against different antibiotics like carbapenems group that's most effective antimicrobial agents for the treatment of infections caused by multidrug resistant bacteria (MDR) (4,5,6).
fusA gene is a housekeeping gene in A. baumannii , encodes for the elongation factor EF-G that catalyzes the GTP-dependent ribosomal translocation step during translation elongation by catalyzing the translocation of peptidyl-RNA from the A to P site of A. baumannii ribosome.During this step, the ribosome changes from the pretranslocational to the post-translocational state as the newly formed A-site-bound peptidyl-tRNA and P-site-bound deacylated tRNA move to the P and E sites, respectively Catalyzing process of the coordinated movement of the two tRNA molecules, the mRNA and conformational changes in the ribosome required of product of this gene based on the importance of fusA gene in translation regulation (7,8,9).So, this study aim at detecting genetic variation and genotype of fusA gene in clinical isolates of these bacteria.

Materials and Methods: Samples collection
In this study two hundred clinical samples were collected (were 50 samples from urine, 50 samples from wound, 50 samples from sputum, 50 samples from otitis) in some hospitals (AL-Kindy Teaching Hospitals, Imam Ali Hospitals, AL-Sader Hospitals) in Baghdad during the period from September into December 2016.

Isolation and identification
The samples were cultured onto MacConkey agar and incubated for 18-24 hrs at 37 o C. The non lactose fermenting isolates were cultured onto CHRO Magar TM medium and incubated for 18-24 at 37 o C, Acinetobacter appears as a red colonies after the incubation period.The isolates were tested by morphologic characteristics and standard biochemical tests according to MacFaddin, (2000) (10).Then confirmation of Acinetobacter spp.isolates was carried out by VITEK-2 Compact system to identification Acinetobacter isolates to species level according to manufactures' instructions (Biomerieux/ France).

DNA extraction and PCR assay
DNA of all twenty A. baumannii isolates from two hundred clinical samples was extracted by wizard® genomic DNA purification kit (Promega, USA) according to manufactures' instructions.Amplification of the fusA gene was performed with specific primers, (Table 1).The reaction program of PCR (initial denaturation at 94 ºC for 2 minutes, and 30 cycles of denaturation at 94ºC for 30 second, annealing at 50ºC for 30 second, extension at 72ºC for 30 second and a final extension at72ºC for 5 minutes) was done.A molecular marker (promega/ USA effective size range: 100 to 1500 bp) was used to assess PCR product size.

Sequencing of fusA gene
Sequencing of fusA gene of all twenty isolates was carried out by sending PCR products of amplification fusA gene to macrogen corporation-Korea using ABI3730XL, automated DNA sequencer.

Genetic Analyses of fusA gene
Phylogenetic study was carried outing which the results were analyzed by using Geneious 9 software.The sequenced DNA were analyzed by BLASTn tool of NCBI GenBanK database.Then comparing was done between the query sequences and those documented in the GenBanK database.The confirmation of bacterial isolates was carried out which the closest alignment match has a very high identity to the homologues in Gene-Bank.The sequencing of target gene that identities were also computed using the pairwise alignment by Geneious 9 software.Multiple sequence alignments were performed using Geneious alignment and the phylogenetic analyses were done by the maximum likelihood method.

Results and Discussion: Isolation and identification
In this study collection of two hundred clinical samples (fifty samples from urine, fifty samples from wound, fifty samples from sputum and fifty samples from otitis) was done.Then laboratory diagnosis of these samples carried out by routine methods and confirmation by using VITEK-2 Compact system.There were twenty confirmed isolates of A. baumannii in all these samples in percentage (10%) included 11 isolates from urine , 4 isolates from wound , 3 from sputum and 2 from otitis.In locally study carried out by Adnan et al.
(2014) (12) the percentage of infection with this bacteria was (10.3%) in different clinical samples.Another locally study by Mosafer, 2007 (13) isolated Acinetobacter baumannii from different clinical sources with different percentage of infection.Distribution of infection with this bacteria in different region in worldwide because this bacterium an important nosocomial pathogens and has different virulence factors confirmed the local rate of infections (14).

Detection of fusA gene
Detection of fusA gene in all twenty isolates carried out to investigate this gene.The results showed that all isolates were positive to presence of this gene as shown in figure (1).

Sequencing of fusA gene
Analyses of sequencing fusA gene of all isolates were carried out by BLASTn tool of NCBI GenBanK database and results of sequencing of all isolates were analyzed by using Geneious 9 software to draw phylogenetic tree among locally isolates alone and among local and global isolates that documented in gene bank.

Genotyping of fusA gene in locally isolates
Phylogenetic study of the fusA gene was done for all (20) clinical isolates A. baumannii from all sources of isolation included urine, wound, sputum and otitis infection.The results in phylogenetic tree found 4 groups of isolates as shown in Figure (2) and Table (2).The isolates in group a included urine and wound sources.Group B included wound and sputum sources.Group C contain otitis and urine sources, the group D included urine, otitis infection and wound sources.These bacteria consider a high virulence pathogen that led to spreading of epidemiology of infections produced by this bacteria and increasing multidrug resistance.Drug resistance in Acinetobacter baumannii is mainly caused by the emergence and produced of extended spectrum betalactamase, fluoroquinolone resistance and the prevalence of multidrug resistance and carbapenem-resistant strains.This problem shows that data in the world organization (National Healthcare Safety Network) in united states centers for disease control and prevention (CDC), in which there are high rates of carbapenem resistance Acinetobacter baumannii throughout the USA and increased nosocomial infections (15,16,17).All that leads to the pathogenesis of these bacteria.The study of housekeeping gene like fusA gene in clinical isolates is very important tool in identification of distribution A. baumannii (18).So phylogenetic tree of this gene was done in this study to understand the epidemiology of Acinetobacter baumannii in Baghdad.

Genotyping of fusA gene in locally isolates comparison with global isolates in GenBank
For studying genotyping of fusA gene in locally isolated Acinetobacter baumannii and comparison with standard isolates.All locally isolates compared with eight standard global isolates documented in gene bank under accession numbers were (CP014528, CP007712, CP006768, CP012006, LT605059, CP003847, CP009256, CP018677).The results in phylogenetic tree (Figure 3) show that there are four groups of isolates.The first group A included locally isolates (AE_22, AE_26) and group E (AE_35, AE_32, AE_33) were not identity with global isolates.The second group B included locally isolates (AE_23, AE_36, AE_37, AE_21, AE_25, AE_27, AE_28, AE_30, AE_39) similarity with global isolates (CP014528, CP007712).The locally isolates in third group C (AE_40, AE_24, AE_31) were identified with global isolates (CP006768, CP012006, LT605059).Group D included locally isolates that were (AE_29, AE_38) and global isolates were (CP003847, CP009256, CP018677).These results in phylogenetic tree Figure (3) showed each group including local isolates and global isolates except group A (AE_22, AE_26), in another meaning each group from the locally isolates identity with specific global isolates except group A. That's may be due to found genetic variation between these isolates and global isolates lead to distribution new isolates (AE_22, AE_26) in Baghdad governorate.

Registration locally sequencing of fusA gene in GenBank
The registration sequence of fusA gene from local A. bumanii in national GenBank was done.The local isolate (AE_35) that not identical with global isolates as shown in Figure 3, was selected to the registration.The nucleotides sequence of fusA gene was registered under accession number (LOCUS KY818057) and protein ID "ARV90995.1.

Conclusion:
In genotyping of fusA gene from clinical Acinetobacter baumannii isolates, the results in phylogenetic tree among local isolates found 4 groups of isolates.From the comparison between local and global isolates , it can be concluded that there are four different groups of local isolates and each group identified with certain global isolates except group A (AE_22, AE_26) and group E (AE_35, AE_32, AE_33 ) which may consider new isolates.The nucleotides sequence of fusA gene from isolate (AE_35) was registered in national GenBank under accession number (LOCUS KY818057) and protein ID "ARV90995.1.

Figure 2 .
Figure 2. Unrooted phylogenetic tree of fusA gene within Acinetobacter baumannii isolates structured with the maximum probability method by Geneious 9 software.