Genetic Characterization of Extensive Drug Resistant Acinetobacter Baumannii: an Appalling Impediment

Introduction: Acinetobacter baumannii infections are a growing public-health concern. The bacterium’s potentiality to acquire resistance to a number of commonly used antibiotics has turned it into a formidable pathogen. Aims: Molecular characterization of extensive drug resistant (XDR) typing of A. baumannii clinical isolates by polymerase chain reaction. Materials and methods: Thirty XDR A. baumannii were investigated for the presence of genes encoding carbapenemase resistance, biofilm capacity, autoinducer synthase, virulence and surface motility by polymerase chain reaction (PCR). Later, the isolates were typed by plasmid-based replicon (Rep) (PBRT) and trilocus sequence typing. Results: All 30 XDR A. baumannii strains displayed genes related to surface motility, autoinducer synthase, virulence determinant, biofilm related genes except PER, and bap, the frequency of which was 83.3% and 76.6%, respectively. Analysis of rep genes showed highest frequency of rep6 and rep2 genes, with frequency of 75% and 65%, respectively. All XDR A. baumannii strains belonged to SG I (European clone II) group. Conclusions: Our results show the extraordinary plasticity of XDR A. baumannii and suggest that the strains have gained endemicity in our hospital, which could be a great concern in the near future.


INTRODUCTION
Acinetobacter baumannii, famed as an "old friend-new enemy" 1 , has emerged as a successful pathogen causing a broad array of clinical infections in hospital and community acquired settings. The organism possesses intrinsic and acquired resistance to a number of commonly used antibiotics and is thus gradually bequeathing intra and inter-hospital setting. 2 Nevertheless, antibiotic resistance rates can vary according to the country, the individual hospital, and may even depend on biological, epidemiological or methodical factors. 3 As the organism has gained hold of the antibiotic resistance, it is not uncommon to find multidrug-resistant (MDR, resistance to at least three classes of antimicrobials), extensively drug-resistant (XDR, MDR plus resistance to carbapenems), and pan-drug-resistant (PDR, XDR plus resistance to polymyxins) nosocomial isolates that are hard to treat with the currently available drugs. 4 Amongst all these, according to World Health Organization (WHO) carbapenem-resistant A. baumannii (CRAB) strains have emerged as one of the most concerning antibiotic-resistant pathogens among other gram-negative bacteria. 5 Carbapenem-hydrolyzing class D β-lactamases (CH-DLs) are determinants of carbapenem resistance in A. baumannii. Four major plasmid encoded CHDLs genes including bla  , bla  , bla  , and bla OXA-51-like have been documented globally. 6 Amongst them, strains carrying bla OXA-58 stand out as the most common type from Europe, Argentina, Australia, the United States and many Asian countries. 7 One of the major factors contributing to drug resistance in A. baumannii associated infections is its biofilm development capacity. 8 Quorum sensing (QS) (autoinducerreceptor mechanism) plays a role in biofilm formation in Acinetobacter baumannii associated infections, though its role in the regulation of other virulence factors is yet to be established. 9 Several published studies have explored the relationship between biofilm and antibiotic resistance in A. baumannii. [10][11][12][13] In this regard, A. baumannii is among the leading nosocomial pathogens with a capacity to colonize venous catheters (CVCs) and cause lower respiratory tract infections (due to contaminated ventilators). 14 Bacteria in the biofilm structure are enclosed in a matrix that increases drug resistance and causes chronic and persistent diseases that are difficult to treat. 15,16 Research conducted on biofilm-associated operon in A. baumannii suggested CsuA/BABCDE-mediated pili formation, which plays a role in the initial steps of biofilm by allowing bacterial cells to adhere to abiotic surfaces resulting in the initiation of microcolony formation that precedes the full development of biofilm structures. CsuE codes for the tip adhesion and its inactivation result in the abolition of pili production as well as biofilm formation. 17 The expression of this operon has been found to be regulated by a two-component system comprising sensor kinase encoded by bfmS and a response regulator bfmR. 18 BfmSR controls the production of capsular exopolysaccharides as well as pilus assembly, and consequently, cell attachment and biofilm formation. 19 A. baumannii also contains pgaABCD locus that encodes a protein which synthesizes cell-associated poly-β-(1-6)-Nacetyl glucosamine (PNAG). 20 In a variety of gram-negative bacteria, it has been demonstrated that biofilm development may depend on N-acyl-homoserine lactone (AHL) signalling molecules. 21 Mass spectrometry has identified AHL signals directed by protein AbaI. The abaI gene is activated in a positive-feedback loop by an AbaI-dependent AHL signal(s). 21 In addition, a homolog of a staphylococcal biofilm-associated protein (Bap) has been characterized in A. baumannii, where it appears to act as an extracellular adhesin and play a key role in biofilm production in A. baumannii. 22 blaPER-1 and Bap, in addition to being involved in biofilm formation, are also involved in the bacterial attachment to human epithelial cells and abiotic surfaces. 23 The formation of pellicles, a specific form of biofilm, occurs at the air-liquid interface and is distinct from submerged biofilms. A correlation between surface associated motility and pellicle biofilm formation has been described for A. baumannii. 24 The organism encodes a diverse range of secretion systems. The type I secretion system (T1SS) is a tripartite system, delivering proteins from the cytosol to the extracellular environment. Interestingly, the activity of the T1SS was shown to have a direct impact on the type VI secretion system (T6SS), suggestive of cross talk between these systems. 25 Among various virulence factors possessed by A. baumannii, outer membrane protein A (OmpA, previously Omp38) is the most abundant A. baumannii OMP and one of the most well-characterized virulence factors. 26 Despite the progress in the study of antibiotic resistance mechanisms in A. baumannii, a more recondite work is knowledge about the genetic factors that have driven the recent evolution of A. baumannii toward multidrug resistance. 27 Sequence analysis of plasmid replicons corresponding to A. baumannii clinical strains has revealed many differences with those from other bacterial species, strongly suggesting that A. baumannii contains its own plasmid types. 28 Research on the genome content of A. baumannii species reveals the specificity of its plasmids and thus, plasmid typing of A. baumannii according to their replicase (Rep) proteins was suggested as an effective tool. 28 Plasmidbased replicon (Rep) (PBRT) typing scheme furnished that there are 19 homology groups (GR1-GR19) based on their nucleotide sequence similarities. 27,29 PBRT method is particularly useful when investigating specific features such as an antibiotic resistance gene. Intriguingly, it will be a very useful method to detect and study these plasmids and to further understand the evolution of resistance. 27 A multilocus sequence-typing scheme or the "trilocus sequence-based typing" (3LST), based on housekeeping genes, for A. baumannii examined the sequence variation within three genes likely to be under selective pressure. This approach, which has the potential for greater discrimination, comprises of three genes: ompA, csuE and bla OXA-51-like .
Outer-membrane protein A (encoded by ompA) is a porin found to induce apoptosis of epithelial cells. The csuE gene codes for part of a pilus assembly system and is essential for biofilm formation. 30 The bla OXA-51-like gene codes for the intrinsic carbapenemase found in A. baumannii. 30,31 The 3LST-based multiplex-PCR assay's purpose was to rapidly assign isolates to profile groups (G), consisting of G1, corresponding to ICII; G2, to ICI; and G3, to ICIII. 30 Subsequently, several studies using this PCR-based typing technique reported the identification of new amplicon combinations, in addition to those expected for ICI-III clones. 31

AIM
The present study determined I) the correlation between the ability of A. baumannii for biofilm formation and distribution of biofilm related genes, and II) carbapenem resistance genotypes in the XDR A. baumannii clinical isolates. Furthermore, we applied PBRT and 3LST schemes to type the isolates according to their replicase and housekeeping genes, respectively.

Bacterial isolates
We performed this study on 30 XDR A. baumannii strains isolated from clinical specimens obtained from various patients admitted in Sina Hospital, a University based Teaching and Research Center (Tabriz, Iran) as a routine bacteriological procedure. The A. baumannii isolates were initially identified by conventional phenotypic methods 32 and later confirmed genotypically (bla Oxa-51 and rpo genes). 33,34 Acinetobacter baumannii ATCC19606 was used as a standard strain.

Detection of blaOXA genes
The bacterial genomic DNA was extracted from overnight cultures of A. baumannii isolates using a DNA purification kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Multiplex-PCR was performed to investigate the presence of carbapenemase genes: bla OXA-143 , bla OXA-23 , bla OXA-24 , bla OXA-51 , bla OXA-58 using primers and amplification conditions as described previously. 39 The primers used are listed in the Table 1.

Quantitative biofilm formation assay
Clinical XDR A. baumannii isolates were analyzed for their ability to produce biofilm using microtiter plate method based on the crystal violet staining method. 40 Briefly, isolates were initially cultured in the Tryptic Soy Broth (TSB) medium comprising 1% glucose. After incubation for 24 hours at 37°C, 20 µl of fresh bacteria containing medium was transferred to the 96-well polystyrene microtiter well containing 180 µl of fresh TSB medium. After incubation at 37°C for 24 hours, the medium was discarded and the adherent cells washed twice with the phosphate-buffered saline (PBS) (pH7.4) followed by addition of 250 µl (99%) methanol and then 200 µl (0.1%) Crystal Violet. The stain was eluted from the adherent cells using 160 µl acetic acid (33%). Wells containing no bacteria were used as controls. Absorbance (optical density) of the eluted solvent was measured for the clinical isolates (ODi) and negative control (ODc) at 570 nm using an UV visible spectrophotometer (Epoch, Biotek). The assay was repeated at least three times using fresh samples each time. The results were interpreted as follows: if ODi < ODc, the bacteria were non-adherent; if ODc < ODi_2×ODc, the bacteria were weakly adherent; if 2×ODc < ODi ≤4×ODc, the bacteria were moderately adherent; and if 4×ODc < ODi, the bacteria were strongly adherent. 40 Detection of biofilm (bap, PER, bfmSR, csuE, pgaA, and pgaD), motility (type I fimbriae, PilT), autoinducer synthase (abaI) and virulence related genes (omp) The bacterial genomic DNA was extracted as stated above according to manufacturer's protocol. Amplification of biofilm associated, autoinducer synthase, virulence and surface motility-related genes was performed using specific primers (listed in Table 1) and PCR conditions in XDR Acinetobacter baumannii isolates. 41,42

A. baumannii sequence-based typing method
Two sets of multiplex-PCR (SG1 and SG2) were performed with primers targeting ompA, csuE and bla OXA-51-like specific alleles. 30 PCR program included an initial denaturation at 94°C for 3 min followed by 30 cycles of 45 s denaturation at 94°C, 45 s primer annealing at 57°C, 1 min extension at 72°C; and a final elongation step of 72°C for 5 min. 30

A. baumannii PCR-based replicon typing (AB-PBRT) method
The bacterial genomic DNA was extracted from overnight cultures of A. baumannii isolates in LB broth using a DNA purification kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. The primers used for AB-PBRT  (39) are listed in Table 2. The PCR amplifications for 19 rep genes were grouped as 11 simplex PCR or as duplex PCR ( Table 2). PCR amplifications were performed as follows: 1 cycle of denaturation at 94°C for 7 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing (mentioned in Table 2) and elongation at 72°C for 1.5 min. The amplification was ended with an extension program at 72°C for 5 min.

Statistical analysis
As all thirty A. baumannii isolates were positive for almost all the genes studied for biofilm, autoinducer synthase, virulence and surface motility-related, thus only the frequencies were calculated as percentages. The association between the genes involved in biofilm formation and the amount of biofilm mass with antibiotic resistant phenoty-

Antibiotic susceptibility testing
On disk diffusion assay, all isolates showed no zone of inhibition around the following disks: ceftazidime, cefepime, amikacin, gentamicin, ceftriaxone, trimethoprim-sulphamethoxazole, ciprofloxacin, ampicillin-sulbactam, imipenem and meropenem. MIC of imipenem and meropenem was >32 µg/ml. MIC of colistin ranged from 0.125 to 2 µg/ ml with MIC 50 and MIC 90 being 0.5 and 1 respectively, thus no isolate was found resistant to colistin. MIC of tigecycline also ranged from 0.125 to 2 µg/ml with MIC 50 and MIC 90 being 0.5 and 2, respectively. Thus, all 30 isolates were XDR.

Detection of biofilm, motility, autoinducer synthase and virulence genes by PCR
Among 30 A. baumannii isolates, all were positive for biofilm-associated and autoinducer genes comprising bfmSR, csuE, pgaA, abaI and pgaD. However, detection rates of bap and bla-PER1 were 76.6% (n=23) and 83.3% (n=25), respectively. However, the mean for biofilm biomass in bap, and blaPER-1 positive isolates were 0.06 nm to 0.37 nm and 0.06 to 0.51 nm respectively. All isolates were also positive for type I fimbriae, PilT motility related genes, and ompA virulence gene.

Tri-locus sequence typing
Sequence-based typing revealed all isolates shared the same combination of alleles at the three loci belonged to only one group 1 (European clone II). No isolate belonged to Group 2 or Group 3 or any new variant.

DISCUSSION
Initially, A. baumannii emerged as an opportunistic pathogen and the treatment of infection was managed with β-lactams. The persistent presence of A. baumannii in the hospital setting allowed it to encounter antibiotics which lead to the emergence of successful clones with particular antibiotic resistance characteristics. 43 Currently, A. baumannii marks the culmination of organisms facing antibiotic resistance, the so-called paralyzing situation in therapeutics. The organism is notorious for causing serious infections in intensive care units (ICUs). 44 In our study, 73.3% of clinical isolates were obtained from ICUs. Unfortunately, all isolates were resistant to antibiotics commonly used by infectious specialist in our hospital setting except colistin and tigecycline. Earlier research studies displays this organism as MDR 45 while, later published research studies marked them as XDR 46 or PDR. 47 More than 90% clinical isolates were resistant to ceftazidime, cefotaxime, cefepime, amikacin, ciprofloxacin, piperacillin-tazobactam, ampicillin-sulbactam and co-tri-Folia Medica I 2021 I Vol. 63 I No. 5  moxazole in an Iranian study performed on clinical isolates obtained from two hospitals. 44 Carbapenem was once considered as an effective drug for the treatment of infections caused by A. baumannii, but in recent decades, the rate of carbapenem-resistant A. baumannii strains has increased dramatically and is spreading throughout the world. [48][49][50][51] Studies from Asian countries including China, Thailand, and Taiwan 52-54 , show carbapenem resistance to vary from 40 to 60%. In general, carbapenem-hydrolyzing class D β-lactamases (CHDLs) in A. bau-mannii is mainly mediated by bla  , and bla OXA-51-like . 55 bla OXA-23 are the most common OXA type of carbapenem resistance in Iran. 56 In the present investigation performed on XDR strains, all isolates had bla OXA-51-like gene while the frequency of bla OXA-23 and bla OXA-24 was 90% and 46.6%, respectively. We did not find presence of bla OXA-58 and bla OXA-143 genes in any isolate. In most studies performed on CHDLs, bla OXA-58 gene has not been found 57,58 while, bla OXA-23 has been reported as an abundant CHDLs, with a frequency of over 80%. 12,59,60 A study of samples collected from 1991 to 2011 showed that the content of the bla OXA gene in A. baumannii changed from bla  and bla  to bla  . Since 2008, the bla OXA-23 gene has been the most abundant type of OXA among Acinetobacter baumannii species around the world. The study also showed that with the change in OXA content, the strains' ability to bind and form biofilm has decreased. 61 The results of our studies and studies in other parts of the world and in Iran show that this bacterium is resistant to carbapenem. As usage of carbapenem is the last resort treatment strategy, it is of great concern.
According to the results of the present investigation, 13.3% XDR A. baumannii strains had potentiality to form strong biofilm while other strains produced either moderate (23.3%) or weak (63.3%) biofilm. No significant relationship was observed between resistance and biofilm formation among A. baumannii species in our study. Contrary to the phenotypic findings, we could observe presence of biofilm genes in all A. baumannii isolates except bap and blaPER genes, which were not present in all strains. Study conducted in China 10 did not indicate a direct relationship between biofilm formation and increased antibiotic resistance, but could show a balance between the two so that weak and strong biofilm strains had the same resistance level. In addition, biofilm production was proposed as a mechanism for survival in strains that have less resistance. Another study also found no difference in biofilm formation in MDR and non-MDR strains. 61 By contrast, a strong association between biofilm formation and drug resistance has been reported in an Iranian study. 62 An Iranian study indicated high prevalence of ompA, csuE and bfmSR genes in XDR strains. 63 Compatible to another studies 62,64,65 we found the frequency of ompA and csuE genes to be much greater than bap gene. All A. baumannii strains harboured pgaD and abaI genes in contrast to another study conducted in Iran, where quiet low frequency of pgaD and abaI genes were reported (45.3% and 14%, respectively). This difference may be due to XDR strains in our study. Studies have shown that the abaI gene, an autoinducer synthase, is involved in the late stages of biofilm formation, and mutation of this gene disrupts the biofilm formation process. 14 Since in our research study, all A. baumannii strains were positive for at least one of the biofilm-associated genes thus, abaI may be positive in all strains. In another Iranian study, the abaI gene frequency was 18%. Higher frequency in our study may be due to inclusion of only XDR strains; nevertheless, we did not find any relation between biofilm Folia Medica I 2021 I Vol. 63 I No. 5 Table 3. Comparative result of Tri-locus sequence typing and replicase typing methods for XDR A. baumannii clinical isolates Tri-locus sequence typing and SGs (sequence groups) 20  +  ------+  -AB 21  +  --+  -+  ---AB 22  +  --+  -+  +  +  -AB 23  +  ----+  -+  -AB 24  +  ----+  -+  +   AB 25  +  --+  -+  -+  -AB 26  +  -+  -+  --+  +   AB 27  +  --+  -+  --+   AB 28  +  --+  -+  -+  -AB 29  +  --+  ---+  +   AB 30  +  --------production and abaI gene. In the present investigation, all A. baumannii isolates were XDR and biofilm producers, which is a clinical apprehension. The frequency of abaI gene was 59.8% in a Chinese study but the isolates were not specifically XDR. 66 In a study performed in Iraq, 66% non-XDR isolates revealed presence of abaI gene and all these isolates were biofilm producer. 67 Based on the results of this study and other investigations, we can conclude that though abaI gene is related to autoinducer synthesis, it plays an important role in biofilm production and is associated with antibiotic resistance. Despite progress in the antibiotic resistance mechanisms in A. baumannii, still knowledge is scarce in understanding the genetic factors that have driven the recent evolution of A. baumannii toward multidrug resistance. 27 A. baumannii may develop resistance to carbapenems through plasmidmediated acquisition of carbapenem-hydrolyzing class D β-lactamases (CHDLs). In particular the bla OXA-58 and bla OXA-23 genes, encoding the OXA-58 and OXA-23 CH-DLs respectively, have been reported from A. baumannii isolates collected from distant parts of the world in association with plasmids. 68,69 In the present study bla OXA-51 like was present in all A. baumannii CHDLs producing strains and bla  was observed in 90% strains while, the bla  was not detected in any isolate.

Replicase typing and replicase GR (groups) and genes
In the present study, XDR A. baumannii were investigat-ed by plasmid replicon typing and more than 60% A. baumannii strains belonged to rep6 and rep2 types. This suggests diffusion of the carbapenem-hydrolyzing oxacillinase genes bla OXA-23 and bla OXA-51 like , known to be the sources of resistance to carbapenems in A. baumannii strains, are related to two multiple plasmid types. Compatible results were obtained on 3LST multiplex PCR typing, where all A. baumannii strains belonged to sequence group SG1, corresponding to international clone II. Studies reported from Spain and Greece shows 100% and 99.4% A. baumannii isolates belonged to international clone II, respectively. 30,70 The results of our research also indicate that SG1 is more common among other groups. Recent study have shown that plasmids carrying these genes may be due to the high prevalence of carbapenem resistance through CHDLs. 27 Prior to our study, Acinetobacter baumannii typing had not been performed using replicase typing in Iran. In a study conducted in Italy, A. baumannii strains belonged to groups 6 and 2, with a frequency of 96.8% and 70%, respectively. Also, the results of these studies have shown that rep 6 may play a major role in the horizontal transmission of resistance among members of this species. 71 Because we did not perform transferability experiment, the genetic dissemination cannot be proved. However, our results showed predominance of rep 6 and rep 2 genes in XDR A. baumannii strains.

CONCLUSIONS
The results of our study shows that we are facing a jeopardous situation. Presence of biofilm associated genes except blaPER-1 and Bap and other virulence genes in all clinical isolates is a feature which confirms the endemicity of A. baumannii and appraises the nosocomial nature of the bacteria. Our investigation showed that all A. baumannii strains belonged to SG1 group and two major replicase groups, which further highlights an emergence of one type of clone and its dissemination. It is thus critical to work on their transfer ability and prevention of resistant bacterial dissemination in order to avoid further restrictions on therapeutic options. In order to evaluate new specific interventions, it is essential to gather specific data on the antibiotic resistance in A. baumannii. As antibiotics are gradually losing their effectiveness today, other ways such as prevention of biofilm formation or using quorum-sensing quenchers should be exploited.