GENE EXPRESSION of blaOXA-51-like and blaOXA-23 in RESPONSE to β-Lactam ANTIBIOTIC IN CLINICALLY ISOLATED ACINETOBACTER BAUMANNII AND ACINETOBACTER LOWFFII FROM URINE SAMPLES

In this study, two isolates A. lowffii, and A. baumannii were obtained clinically, and genomically identified by 16S rRNA assessment with the accession number (MH685113.1 and MH685112.1) for A. lowffii and A. baumannii respectively. The sensitivity profile of the isolates was variable, and A. baumannii was the most resistant strain towards a wide range of antibiotics, and it did not show any growth defect in the presence of β-lactam antibiotic, in comparison with A. lowffii. We identified that the blaOXA-23 gene was responsible for imipenem resistance in A. baumannii, whereas, blaOXA-51-like was moderately confer resistance towards A. lowffii which lack of blaOXA-23. This was determined when the isolates were subjected to qRTPCR. We identified that the blaOXA-23 gene was increased about 1-fold in the presence of imipenem, whereas, blaOXA-51-like did not increased in comparison to the control. Bioinformatic analyses revealed that blaOXA-23 is located in the cytoplasm, and blaOXA-like 51 is located in the periplasm. Our hypothesis suggests that blaOXA-23 gene have a major contribution in the outbreak of multidrug resistance Acinetobacter species.


INTRODUCTION
Acinetobacter spp. is a non fastidious Gramnegative bacteria, belongs to gamma protobacteria, nonfermenting, stringency aerobic and non motile. It was known to be positive to catalyse and negative to oxidase, in addition to the high GC contents (29). The genus Acinetobacter comprises of 31 well known published species that predominantly exist in nature and mainly associated with nosocomial infection, urinary tract infection, skin and other soft tissues, and in bloodstream (13) The identification and characterization of Acinetobacter spp by molecular technique like 16S-23S rRNA gene spacer region was more accurate and reliable, in addition to the method based on rpoB gene sequence (5,28,30). A. lowffii, amongst the Acinetobacter spp that generally isolated from blood, and may associated with bacteremia, which resulted from skin contamination (44). Moreover, A. baumannii is another species which is mainly associated with nosocomial infection and important opportunistic pathogen acquired infection, especially in intensive care unit (54). Interestingly, A. lowffii is considered to be normal flora of some parts in the human bodies like skin, oropharynx and perineum of non infectious human (26).
Recently, resistance of pathogens to multiple classes of antibiotics is of great concern to the scientists. Acinetobacter species has been known for their ingenuin resistance to antibiotics, and also for their ability to acquire genes encoding determinants, and other mechanisms that involved β-lactamases production and aminoglycoside-modifying enzymes (2). Moreover, the presence of multidrugresistance (MDR) Acinetobacter spp. in longterm care facilities and in acute care hospitals has been reported (36). The overcome and persistence of MDR species is patient care unit is due to the ability of patient to get the infection, or to the presence of patients that already colonized or infected with these organisms, and also lack of infection control procedures (2). Nevertheless, Acinetobacter spp. possesses several mechanisms that mediate antibiotic resistance. One of the most potential ways is the antimicrobial inactivating enzymes, which includes a wide spectrum of β-lactamases that degrade and confer resistance to penicillins, cephalosporins and carbapenems, however, cephalosporins can be repelled by chromosomally encoded gene AmpC (55). According to amino acid sequence consensus, these β-lactamases are classified into four major molecular classes; A, B, C and D, where A, C and D are inactivate the βlactam ring via active catalytically serine residue, like AmpC (59). On the other hand, zinc is required for catalyst the activity of βlactamase Class B, which is a metallo-enzyme and exhibits different mechanism of resistance (32). Amongst these enzymes, OXA-51 was found to be involved in carabapenem resistance, when it was characterized form two isolates of A. baumannii clones in Argentina (5). OXA-23 was firstly mentioned in 1995, and belongs to β-lactamase Class D or Carabpenem-hydrolyzing class D β-lactamase (CHDL) that confers resistance towards imipenem and carbapenem. Because it was reported mainly in A. bumannii, it was formally named as ARI-1 (59,60). Moreover, antibiotics can be repelled by efflux pump system, which are able to remove a wide range of antibiotics out of the bacterial cell (2). Furthermore, antibiotic resistance genes can be acquired either from other organisms or from mutant construct which leads to develop the resistance phenotype over time in Acinetobacter spp. In a comparative genomic study of an epidemic and MDR Acinetobacter strains isolated in France, revealed that 45 resistance gene-containing resistance island, were acquired from Pseudomonas, Salmonella and Escherichia genera (17). In this study, we demonstrate that the expression of blaOXAlike 51 and blaOXA-23 is variable in response to imipenem addition in A. baumannii and in A. lowffii. In addition, the ability of these isolates to survive under different concentrations of β-lactam drug was examined. We also determined the localization pattern of these enzymes in Acinetobacter isolates.

MATERIALS AND METHODS Bacterial Isolates Media and Growth Conditions
The bacteria were clinically isolated from urine and identified as A. lowffii, A. baumanni by normal Vitek 2 system (bioMerieux). The isolates were routinely subcultured either on Mackonkey agar and on nutrient agar (NA) plates at 37 ͦ C every 24 hr and under aerobic conditions. For liquid cultures, the isolates were inoculated in Mueller Hinton (MH) broth and incubated aerobically at 37 ͦ C for 24 hr with gentle shaking. Bacterial growth curve was performed either under normal conditions, or with the presence of antibiotic. In both cases, bacterial growth was monitored by measurement of OD 600 against the relevant media as a control (blank). The growth was observed every hour started from OD 600 ≈0.1 to approximately OD6 00 ≈1.5. DNA Manipulation and Isolation, 16S rRNA Sequencing, PCR Genomic DNA from isolates was isolated using Wizard Genomic DNA purification Kit (Promega), and according to manufacturer's instructions. In order to confirm the identification of Acinetobacter species, 16S rRNA sequencing was required. The amplification and sequencing of 16S rRNA was performed according to (33), with some modification. The PCR amplification reaction of interested genes was carried out in a 25µl reaction containing 12.5µl of Green Master Mix, 1ul of 10 pmol/µl of forward and/ or reverse primer (Table 1), 2 µl of genomic DNA template. The volume was completed to 25µl by adding nuclease-free water. PCR reactions were set up as detailed in manufacturers' guideline with a modification as necessary. PCR products were resolved on 1.5 % agarose gel, and visualized by a UV transilluminator and the image was captured by digital camera (Canon, US).

qRT-PCR Analyses
Acinetobacter cultures were grown in triplicate in MH broth under normal aerobic conditions for 24 hr. The growth was adjusted to an OD 600 nm of 0.1 before 20 µg of imipenem was added. The growth was monitored till mid phase (OD 600 nm of 0.5) and the RNA was extracted directly from the cultures using the SV total RNA isolation system (Promega, US) according to the manufacturer instructions. Similar was done with cultures without imipenem as a control. Primers used in RNA extraction are listed in (Table 1).

RESULTS AND DISCUSSION Dendogram of Acinetobacter isolates
Since the 16S rRNA gene was developed as a new standard for identifying bacteria, a wide range of bacteria has been determined by 5S, 16S and 23S rRNA gene sequences (20,56). The comparison of 16S rRNA gene is done among all bacteria and archeobacteria, and within eucaryotes by 18S rRNA gene (10). In general, the 16S rRNA is universal in bacteria, large, about 1,550 bp in length, with variable and conserved regions, and has enough interspecific polymorphisms of 16S rRNA to confer distinguishing measurements. The conserved region at the beginning and at the end of 16S rRNA gene is almost used as a template for primers, whereas, a comparative taxonomy is provided by a variable region sequence (10). The clinically isolated A.baumannii HHR1 possesses 16S rRNA gene with 794 bp, which is then used to generate the dendrogram of phylogenetic tree ( Figure S1). A. lowffii HHR2 is shown to have 1354 bp 16S rRNA gene ( Figure S2). The sequence alignment of entire gene of HHR1 is showed 95 % similarity with the relative isolates available in the NCBI (Figure 1 A), while, the degree level of similarity of A. lowffii HHR2 with the corresponding isolates is about 94 % (Figure 1 B), as it was showed by tree relationship. Using of 16S rRNA gene to distinguish between species was recorded in many studies, where Niesseria meningitides could be tracked by 16S rRNA (50,3) The variability in the length of 16S rRNA initial gene in both isolates is depend on the sequenced region, and in most cases, initial base pairs provide a comparable data in clinical isolates (25), whereas, it was reported that describing a new species required sequencing of entire 16S rRNA gene (48,50). The genetic map of A. baumannii showed that it possesses a robust protection enzyme clusters associated with carbapenems, which so called OXA-type carbapenemases. OXA-23, OXA-24, OXA-58, and OXA-51 among these groups, which might be either chromosomally or plasmid encoded genes, and they are defined according to the basis of sequence comparison (22, 53), and most notably that transferable imipenem resistance can be conferred by OXA-23 (22). To assess the existence of OXA-51 and OXA-23 genes in isolates, blaOXA-51 and blaOXA-23 were PCR amplified using blaOXA51-FWD/blaOXA51-R, blaOXA23-FWD/blaOXA23-R and blaOXA361-FWD/blaOXA361-R primers respectively Table 1), and genomic DNA of A. lowffii and A. baumannii were used as a template. The PCR reactions were used in DNA electrophoresis and the DNA products were electrophoresed and visualized under the UV light (Figure 2 C). Interestingly, the results showed that the bla OXA-like 51 (353 bp) was found in A. lowffii and A. baumannii, whereas, the bla OXA-23 (501 bp) was found only in A. baumannii ( Figure 3C). The bla OXA-51-like is may be a sole carbapenem resistance in A. lowffii and A baumannii only if ISAba1 is located upstream bla OXA-51-like , as the former (ISAba1) provides promoter for this gene ( Figure 2B) (58). Unlike, bla OXA-23 is found only in A. baumannii, and may be chromosomally located and flanked by one or two copies of ISAba1 ( Figure 2A) (11,37). Antibiotic sensitivity phenotype of Acinetobacter isolates In order to investigate the antibiotic susceptibility pattern of A. lowffii HHR1 and A. baumannii HHR2, antibiotics were selected according to their target and mode of action; gentamycine (aminoglycoside), imipenem (βlactum), ciprofloxacime (floroquinolone), Azithromycine (macrolids). In addition, tetracycline, doxycycline and rifampicine were also used in this study. The result showed that the drug resistance pattern of Acinetobacter isolates is highly variable among the species, thus reflect the variation in the mechanism that mediate drug resistance. In (Figure 3), it was clearly obvious that A. baumannii was significantly resistant to the most of antibiotics, compared with A. lowffii which exhibits a remarkable inhibition toward these antibiotics. In addition, A. baumannii was completely resistant to trimethoprim, aminoglycoside, floroquinolone, samacyclin and doxycline, and slightly sensitive to tetracycline and macrolide, whereas, A. lowffii was susceptible to these drugs. However, both strains were resistant to rifampicin. Moreover, A. lowffii exhibits a moderate resistance toward floroqunolone, samacycline, tetracycline and macrolide. The β-lactam antibiotic (imipenem) was able to inhibit the growth of A. lowffii compared to A. baumannii, where the former showed a significant inhibition (85 mm), and also slightly attenuate the viability of A. baumannii. The majority of Acinetobacter spp. is considered as a multi-drug resistance which due to their ability to acquire genes encoding resistance determinants, as well as up-regulation of efflux pump compartments and enzyme-target modification (18). The susceptibility of Acinetobacter species towards floroquinolone (ciprofloxacime) was variable, where A. baumannii exert a complete resistance. Like other Gram-negative bacteria, Acenitobacter acquired resistance to floroquinolones by point mutations in DNA gyrase or DNA topoisomerase IV. The amino acid substitution of gyrA (Ser83 to Leu 83) and parC (Ser80) have been reported to decrease the binding with the drug (53). Mutation occurs firstly in gyrA and accompanied by mutation in parC, and thus suggests that gyrA is the target of floroqunolone (6). Aminoglycosides (gentamycine) were also exhibit a fluctuated effect on A. lowffii compared to A. baumannii. The resistance mechanism of A. baumannii to gentamycine is performed by efflux pump, which play an important role to extrude these compounds out of the cells (13,31,49). Among different antibiotics, imipenem is the most effective antibiotics against A. lowffii, whereas, A.baumannii was slightly inhibited. This is due to the ability of A. baumannii to produce class D β-lactamases like bla OXA-51 , bla OXA-58 and bla OXA-24 (33).The gene encoding oxacillinases (OXA) have been reported in several Acinetobacter species, where bla OXA-51 like gene is the naturally occurring gene in A. baumannii and contributes in resistance mechanism. Noteworthy that the serine carbapenemase (bla OXA-23 ) was firstly identified in A. baumannii isolated from blood culture in Eidenbergh (16).

Figure 3. Antibiotic resistance profile of
Acinetobacter isolates Overnight cultures of A. lowffii HHR2 and A. baumannii HHR1were diluted up to 10 -5 cfu/ ml. A 100 µl was spread out on MH agar plates by a sterile cotton swab, and prepared antibiotic discs were placed on the plates with equal distance. The plates were incubated aerobically at 37 ͦ C for 24 hr, and the zone of inhibition was calculated. The black arrow means that there is no inhibition zone. Error bars represent the standard deviation from the mean of three independent biological replicates.

Effect of β-lactam imipenem on the Viability of Acinetobacter isolates
Among all examined antibiotics, Acinetobacter isolates exhibit variable response to imipenem, so imipenem was selected to determine its effect on the viability of Acinetobacter isolates, the growth of all strains was monitored in the presence of different concentrations of imipenem ( Figure  4), and the results showed that A. lowffii HHR2 was strongly inhibited by imipenem , particularly at 30 µg ml -1 after 9 hr incubation ( Figure 4B). In contrast, A. baumannii HHR1 was able to survive under different concentrations ranged from 10-30 µg ml -1 ( Figure 4C). However, it is worth noted that the growth of both isolates were comparable and reached to the same OD 600 after 20 hrs incubation ( Figure 4A). Till to date, there is no evidence that carbapenem resistance profile in A. lowffii is acquired by OXA-23 or OXA-51, however, OXA-51 is a natural occurring CHDL and OXA-143 is intrinsic to A. lowffii (16). Carbapenem/Imipenem resistance can be acquired by several mechanisms, such as decrease the affinity of pencilline-binding proteins to carbapenem/ imipenem, decrease the permeability of the outer membrane, repel of β-lactam drugs, as well as β-lactamases activity.
Although, most of carbapenem/imipenem resistance in bacteria is attributed to class B β-lactamases, the combination of over-expression of β-lactamase especially AmpC β-lactamase, and reduced permeability, will overcome the resistance to carbapenems (61). To some extent, many researchers stated that OXA-51 was characterized from two clones of imipenemresistance A. baumannii in Argentina (4). High level resistance pattern toward imipenem and meropenem was also observed in nosocomial A. baumannii isolates harbouring OXA-51 with MIC >> 8 mg ml -1 (21,39). Moreover, the south East clone in the United Kingdom and T clones from hospital in Midland region are associated with only bla OXA-51 and carbapenem resistance. In contrast, two clones (OXA-23-1, OXA-23-2) have been detected in hospitals in the United Kingdom, were show resistant pattern toward imipenem and meropenem, and both possess bla OXA-51-like along with bla OXA-23 (4). Similarly, (60) reported that the clones of A. baumannii isolated from United Kingdom hospital are resistance to carbapenem only if ISAba1 is located adjacent to bla OXA-51-like , whereas, others with no ISAba1 are susceptible. In the mean time, the clones producing bla OXA-23 and having ISAba1 were consistently resistance. In another study also confirm that the insertion sequences ISAbA1 and ISAbA3 provide strong promoter to bla OXA-23 and bla OXA-58 genes respectively, when it is located to the upstream region for both genes and increase the production of the genes, thereby increase the resistance to carbapenems (20). A question is raised up whether the resistance to imipenem is due to bla OXA-51-like or bla OXA-23 or both, by taking the above findings stated that A. baumannii HHR 1 is more resistance to imipenem than that of A. lowffii HHR2 where the later have only OXA-51 carbapenemase. To unequally evacuate the mechanism mediated resistance to imipenem, (50) revealed that OXA-50, OXA-51 and OXA-60, as a part of OXA-type carbapenamases, are naturally occurring enzymes in A, baumannii, Psuedomonas aeruginosa and Riktsia pickettii, however, some A. baumannii strains lack bla OXA-51 family genes, and (22,9) clearly showed the ability of OXA-51 to resist imipenem if linkage with ISAba1. Furthermore, (27) documented that strains harboring bla OXA-23 exhibit imipenem MICs one to two dilution higher than that of those not carrying bla OXA-23. In A. lowffii, the genomic DNA clearly revealed that it producing bla OXA-51-like gene but it was so susceptible to imipenem, even the gene is flanked by ISAba1, and A. baumannii harbouring both genes with ISAba1. Our results conclude that the presence of bla OXA-51like gene in all isolates did not mainly confer resistance to imipenem, whereas, bla OXA-23 like gene in A. baumannii clearly refers to the major contribution to imipenem resistance.

Gene-Expression analyses of bla OXA-23 in A. baumannii HHR1 and A. lowffii HHR2
Hence, the imipenem was able to significantly reduce the viability of A. lowffii HHR2, and with no effect on the A. baumannii HHR1 growth, qRT-PCR was performed to evaluate the expression of bla OXA-51-like and bla OXA-23 genes in response to imipenem, however, up regulated of β-lactamases is not critically compromised the increase/ decrease the level of carbapenem resistance to its substrate. RNA was extracted from A. lowffii HHR2 and A. baumannii HHR1during mid-log growth under two conditions (-/+ imipenem). The primers were used to amplify both bla  and bla OXA-51-like in the presence of 20 µg impenem ( Figure 5A, B). The expression of bla OXA-51-like with imipenem is shown relative or less to the expression levels normalized to 1.0-fold under normal condition in both strains ( Figure 5 C, D),whereas, the expression of bla OXA-23 was increased to 1.0-fold compared to the control ( Figure 5C). These results confirmed the vital role of bla OXA-23 in Blactam antibiotics. The identification of Acinetobacter species that particularly isolated from patients is vital and essential. Molecular identification methods such as DNA_DNA hybridization, 16S rRNA gene sequence were widely used as an efficient way to identify bacteria (34). The nosocomial infection is attributed to A. baumannii which develop a robust system to resists wide spectrum of antibiotics worldwide, especially carabpenem and emipenem (19,38). Generally, bla OXA-51 like gene is weakly expressed, unless, it may up regulated by insertion sequence ISAba1that supply a promoter sequence to increase the copy gene number by change the location from chromosome to a plasmid (7,58). It was reported that OXA-51-like β-lactamse could be deleterious to A. baumannii and attenuate the resistance pattern, whereas, the OXA-51-type enzymes forms a large group of CHDLs with significant role in protection (50). Unlike, the plasmid-born bla OXA-23 , bla OXA-58 genes are highly up-regulated and associated with ISAba1, ISAba2, ISAba3 or IS18, and thus lead to high resistance capacity to carbapenems (43). According to the expression pattern of OXA-23, we hypothesized that imipenem is act as a signal to promote the expression of bla OXA-23 by ISAba1copy in A. baumannii, but it does not be a good inducer for OXA-51 neither in A. baumannii nor in A. lowffii.

Localization of blaOXA proteins in the cell envelope of Acinetobacter isolates
In order to unequivocally determine the localization of blaOXA proteins, Acinetobacter isolates were grown under aerobic conditions with 20 µg imipenem and fractionated into periplasm and cytoplasm fractions, similar assay was performed without imipenem as a control. All fractions were subjected into 12 % SDS-PAGE. The SDS-PAGE (Figure 7) of the cytoplasm fractions showed that blaOXA-51 is located in the cytoplasm and the molecular weight is around 30 kDa in A. lowffii HHR2 and A. baumannii HHR2 ( Figure 7A, C), and it did not show any expression pattern according to imipenem addition. This is clearly indicated that blaOXA-like 51 have no potential role in Blactam resistance. On the other hand, blaOXA-23 protein is shown to be located in the periplasm with the molecular weight around 30 kDa in A. baumannii HHR1 and it is absence in the PM fraction of A. lowffii HHR2 ( Figure 7B, D). Most notably that blaOXA-23 is slightly exhibits a pattern of expression when A. baumannii HHR1 was grown under imipenem stress (Figure 7 D). These results are clearly revealed that blaOXA-like51 is located in the cytoplasm and blaOXA-23 is a periplasmic protein with putative signal sequence, and have an indispensable role in βlactam drug resistance. These findings are consistence with qRT-PCR results.

Figure 7. SDS-PAGE of cytoplasm and PM fractions of Acinetobacter isolates.
Acinetobacter isolates were grown in MH broth media under aerobic conditions to an OD 600 of 0.3, and 20 µg of imipenem was added to the strains. The strains were incubated for 24 hr before being fractionated into cytoplasm and periplasm which was prepared by osmotic shock. Control is represented by fractions without imipenem. The fractions were mixed with 1X protein loading dye, and subjected into 12 % SDS-PAGE. (A); Cytoplasm fraction of A, lowffii, the red arrow represents the blaOXA-like 51, (B); PM fraction of A. lowffii, (C); the cytoplasm of A. baumannii, the blaOXA-like 51 is represented by thick red arrow, and (D); is refer to the PM of A. baumannii , and the 30 kDa bla OXA-23 is indicated by thick red arrow. Lane-IPM is refers to the PM or cytoplasm fractions in A. baumannii; and A. lowffii with no addition of imipenem (as a control). Lane +IPM; represents the fractions with imipenem addition. Lane M is the PageRuler™ Plus Unstained Rec. Protein Ladder (Promega). ̴ 20 µg of protein was loaded into each well.

Structural modelling of blaOXA-23 protein in A. baumannii HHR1
The predicted structure of blaOXA-23 was modelled by Swiss-Model Server and superimposed with blaOXA225 according to (35), with some differences. The structure of blaOXA-23 in our isolate is seems to be similar to blaOXA-225 with identity of 99.21, whereas, only 77% similarity with blaOXA-24 has been found. The surface structure of blaOXA-23 protein is composed of uncharged residues and that why this protein does not purified properly by ion exchange (57). The 3D crystal structure of blaOXA-23 (Figure 8 A right) revealed that it composed of 6 β-strands running antiparalel at N-terminal, and wrapped by 7 α-helices near the centre, connected with many turns and short β-strand with long turn at the C-terminal. The differences between blaOXA-225 and blaOXA-23 are between (Lys 82 and Asp 62) in the α-helix 1, and between (Pro225 and Ser205) in the loop at the centre of the active site (Figure 8 B). It was noted that OXA-23 has a long loop connecting β5 and β6, composed of A220, D222 and I225, and thus provides a large loop area contributes to the binding affinity to carbapenem. Unlike OXA-23, OXA-24 has G224 homologues to D222, and G218 and V223 which homologues for A220 and I225 respectively in OXA-23 (24). In our study, OXA-23 has A220, D222 and P225 in the loop connecting β5 and β6. The presence of proline in the loop makes a kink that might have a significant role in the binding affinity not only for imipenem, but also for other drugs. Instead, OXA-225 has a Ser205 which may due to alter the position of the loop near the active site. For specificity and activity of blaOXA-23 to binds and hydrolyse imipenem, the amino acid arrangement of both protein seems to be identical and both have as so called the bridge above the active site (Figure 8 A left). The bridge of blaOXA-23 is distinctive and distinguishes it from that of blaOXA-48, which has a wide open active site (12). The bridge of F110 and Met 221 in blaOXA-23 mediates a tight imipenem binding site, and play an important role in resistance mechanism. Note worthy that blaOXA-24 and blaOXA-40 have an identical bridge across the active site, this suggests that all enzymes use the same mechanism with strong enzymesubstrate binding (47,48). In this study, A. lowffii HHR2 and A. baumannii HHR1 were previously isolated and identified by 16S rRNA, and OXA-51 and OXA-23 genes were PCR screened. The efficiency of these genes to resist imipenem was determined by different aspects. Here, we found that OXA-23 has a major contribution in drug resistance than OXA-51, and the former could be over-expressed by IS elements. This expression is enhanced or up-regulated in the presence of several concentrations of imipenem, where OXA-23 was increased by 1fold, whereas, up-regulation of OXA-51 was not detected in A. baumannii. Another approach confirms that A. lowffii is completely inhibited after 24 hr, as OXA-23 is missing.
Interestingly, ribbon structure model of OXA-23 showed it has a distinctive bridge across the active site to facilitate the binding of enzymes with the substrate. The fractionation of both isolates showed that OXA-23 is located in the periplasm and OXA-51 in the cytoplasm, and thus clearly ruled out the significant contribution of OXA-23 in imipenem resistance.