In this study, A. baumannii strains 554S and 554L were isolated from the blood sample of a 10-year-old girl with a burn injury who was hospitalized at Taleghani Burn Hospital in Ahvaz, southwest Iran, on July 25, 2012. On July 27, she became febrile, so a blood culture was realized. Because of the delay between admission and positive culture, we considered that the infection was hospital-acquired. From July 27 to July 31, she was treated empirically with 20 mg of amikacin and 1 g of ceftazidime. Then, on August 1, her antibiotic treatment was replaced with 500 mg of meropenem and 2 g/250 mg of piperacillin-tazobactam. From August 3 to August 11, this treatment was replaced with 500,000 IU of colistin. Then, the patient moved hospitals and sadly died on August 21, 2012. Thus, we believe that the patient eventually died from the burn injury and not from the infection with these bacteria. After the blood sample was plated on chocolate agar, two different colonies with different morphological aspects were observed: one small and one large colony (Fig. 1). The two isolates were identified by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Microflex, Bruker Daltonics, Bremen, Germany) as Acinetobacter baumannii strains. Antibiotic susceptibility testing (AST) was performed by the disc diffusion method and interpreted according to the European Committee on Antimicrobial Susceptibility Testing guidelines (http://www.eucast.org/). The two isolates were resistant to carbapenems but full AST revealed that the strains do not have the same profile. The isolate 554L harboring blaOXA-23 was resistant to all antimicrobials tested with the exception of tigecycline and colistin, whereas the isolate 554S harboring blaOXA-72 was also susceptible to doxycycline and tetracycline (Table 1).
Genomic DNA (gDNA) of A. baumannii strains 554S and 554L were sequenced on a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) using the paired-end strategy. Raw reads were assembled with A5-miseq software. Genomes annotation was performed by Rapid Annotation using Subsystem Technology (RAST). tRNA gene detection was performed using the tRNAScanSE 2.0 tool, whereas ribosomal RNA genes were detected using RNAmmer. The sequence type (ST) of strains was analyzed by the MLST 1.8 server of the Center for Genetic Epidemiology. Antimicrobial resistance gene profiling was performed in silico using ARG-ANNOT. Further bioinformatic analysis, such as identification of genomic islands, prophage sequences, clustered regularly interspaced short palindromic repeat (CRISPR) sequences, secondary metabolite gene clusters and plasmid presence were predicted by employing IslandViewer, PHASTER, CRISPRFinder, antiSMASH and Plasmid finder tools, respectively. The percentage of similarity between strains was calculated by pairwise comparison of their Average Nucleotide Identity based on Blast (ANIb) by Jspecies software.
The draft genome of 554S had 386 scaffolds that comprised 4,056,106 bp with a 39% G + C content. Similarly, the 554L draft genome was composed of 580 scaffolds, having a total size of 3,922,359 bp long with 39.1% G + C content. The draft genomes of A. baumannii strains 554S and 554L contained 3884/3689 coding sequences, 8/8 rRNAs, 64/62 tRNAs, respectively. In silico MLST analysis using the Pasteur Institute typing scheme revealed that strains 554S and 554L belong to ST 307 and ST 2, respectively. The strain 554S had 97.1% of similarity with the strain 554L. ARG-ANNOT result analysis revealed that the genomes of strains 554S and 554L encoded 12 and 16 antimicrobial resistance genes, respectively. Genes that harbored resistance to beta-lactams (blaMBL, blaA1, blaA2), aminoglycosides (armA, aphA6), macrolides (mphE, msrE) and sulfamides (sul2) were detected in both genomes. Moreover, in the 554S genome we found genes blaADC-76, blaOXA-72, blaOXA-235, blaOXA-64, while the genes blaADC-73, blaTEM-1D, blaOXA-23, blaOXA-66, aph3"Ia, strA, strB and tetB were unique to the 554L genome (Table 1). Both strains presented the disrupted ATPase encoding comM gene. The genome of the isolate 554S contained at least 28 genomics islands and one complete prophage sequence. Also, three CRISPR sequences could be predicted. For the genome of the isolate 554L, 23 genomic islands and one incomplete prophage sequence were found but no CRISPR sequences. The presence of three putative secondary metabolite gene clusters, including arylpolyene, siderophore and non-ribosomal peptide synthetase (NRPS) gene clusters, could also be predicted in both genomes whereas hserlactone-arylpolyene was predicted only in the 554L genome. Neither strain carried a plasmid.