INTRODUCTION:

The Acinetobacter species especially, Acinetobacter baumannii is the most important species associated with hospital-acquired infections worldwide. This aerobic Gram-negative coccobacillus had been regarded as a low-grade pathogen, but it is a successful pathogen responsible for opportunistic infections of the skin, bloodstream, urinary tract, and other soft tissues. Because many A. baumannii infections have suddenly been reported among veterans and soldiers who served in Iraq and Afghanistan. A. baumannii is referred to as “Iraqibacter.”

Multidrug-resistant (MDR) A. baumannii has spread to civilian hospitals in part by cross-infection of injured military patients repatriated from war zones. Most A. baumannii infections occur in critically ill patients in the intensive care unit (ICU) setting and account for up to 20% of infections in ICUs worldwide. Furthermore, the frequency of community-acquired A. baumannii infections has been increasing gradually¹.

The persistence of A. baumannii in a hospital environment exposes it to constant selective pressure imposed by antibiotics, which, in addition to its remarkable ability to acquire resistance determinants, has led to the emergence of multidrug-resistant (MDR) A. baumannii. Resistance to carbapenems has been reported worldwide, causing special concern because it is the chosen drug to treat infections caused by A. baumannii². Resistance to β-lactams in A. baumannii is primarily mediated by β-lactamases, overproduction of the intrinsic AmpC cephalosporinase is largely responsible for cephalosporin resistance, recent phylogenetic analysis found that the chromosomal AmpC genes in Acinetobacter spp. probably descended from a common ancestor, these AmpC variants are now designated Acinetobacter-derived cephalosporinases (ADCs). ADCs typically hydrolyze penicillins and narrow- and extended-spectrum cephalosporins but not cefepime or carbapenems, save for one exception, these cephalosporinases are predominantly restricted to the genus Acinetobacter³.

Carbapenem resistance in A. baumannii is mainly mediated by the production of class D β-lactamases (oxacillinases), particularly via the acquisition of genes encoding OXA-23, OXA-24/40, OXA-58, OXA-143, and OXA-51, which are intrinsically located on the chromosome. The blaOXA-23 gene was originally identified on the chromosome of Acinetobacter radioresistens and is currently widespread in A. baumannii, where it is associated with different genetic structures in the chromosome and plasmids².

The present study aimed to determine the presence of CRAB isolates and the prevalence of AmpC genes responsible for cephalosporinase-resistance and the prevalence of blaOXA-23 and blaOXA-235 among clinical isolates of A. baumannii collected from two hospitals in Hillah City.

2. MATERIAL AND METHODS:

2.1 Isolation and Identification of Isolates:

A total of 1100 clinical specimens (included 390 burn swabs, 155 wounds swabs, 50 throat, 400 urine and 105 blood) were collected from patients in Hillah hospitals (Al-Hillah Teaching Hospital and Medical City of Mirjan Hospital) over one year period starting from April, 2016 to February, 2017. Isolates were recovered from clinical samples after culturing on MacConkey agar and incubated for overnight at 37°C, non-lactose fermenting bacteria were sub-cultured and incubated for additional overnights. Suspected bacterial isolates which their cells are Gram negative coccobacillary or diplobacillus and negative to oxidase which further identified using VITEK2 system and confirmed by PCR technique by housekeeping genes.

2.2 Antimicrobial susceptibility testing

Isolates were cultured on Mueller-Hinton agar and their susceptibilities to different antibiotics were tested by disk diffusion method according to the Clinical and Laboratory Standard Institute’s guidelines⁴.

2.3 PCR amplification:

DNA was extracted from the isolates by using genomic extraction mini kit according to the manufacture instructions (Bioneer Company, Korea). To amplify the genes encoding carbapenemases, a monoplex-PCR was run using the primers of AmpC (663bp: F/5′- TAA ACA CCA CAT ATG TTC CG -3'and R/5′- ACT TAC TTC AAC TCG CGA CG -3') were described by Bou and Martinez-Beltran⁵, blaOXA-23 gene (1066 bp:F/5′-AAG CAT GAT GAG CGC AAA G-3'and R/5′-AAA AGG CCC ATT TAT CTC AAA-3') were described by Senkyrikova⁶ and blaOXA-235 gene (750 bp:F/5′-TTG TTG CCT TTA CTT AGT TGC-3'and R/5′-CAA AAT TTT AAG ACG GAT CG-3') were described by Higgins and his colleagues⁷.

Amplification was performed in a 20 μl volume as recommended by Promega Master mix instruction. PCR amplifications were carried out on a thermal cycler (Prime, England). The cycling conditions for amplification were as follows: for AmpC gene, initial denaturation of 94°C for 90 sec and 30 cycles of 30 sec at 94° C, 30 sec at 57° C, and 1 min at 72°C, followed by 10 min at 72°C, for blaOXA-23 gene initial denaturation at 95 C for 2 min and 30 cycles of 30 sec at 95°C, 30 sec at 53°C, and 110 sec at 72°C, followed by 5 min at 72°C for blaOXA-235 gene initial denaturation at 95 C for 2 min and 30 cycles of 30 sec at 95°C, 30 sec at 52°C, and 1 min at 72°C, followed by 5 min at 72°C. Amplified products were detected by agarose gel electrophoresis in 1% Tris-borate-EDTA (TBE) agarose (Promega, USA) and staining with Eco safe stain. The electrophoresis result was detected by using gel documentation system.

3. RESULTS:

The bacterial isolates obtained as a pure and predominant growth from clinical samples were only considered for the present study. Based on morphological and cultural properties of Acinetobacter baumannii (isolates that exhibit small colonies, non-fermented lactose on MacConkey's agar, no hemolysis on blood agar), as well as biochemical characterization. The results revealed that only 17/1100 (1.54%) isolates were belonged to A. baumannii, and the percentage of CRAB isolates were 47% (Table (1).

This study showed that a total of 933/1100(84.81%) isolates were identified as other bacterial spp., and 176/1100 (16%) samples showed no bacterial growth. Identification of A. baumannii isolates was carried out by the standard biochemical tests according to the Bergey's Manual of Determinative Bacteriology^8, ⁹and by Vitek2 system.

Table (1): Distribution of bacterial isolates recovered from clinical samples from two hospitals in Hilla city.

Hospital's name	No. of samples	*No. (%) of Acinetobacter baumannii* isolates**	No. (%) of other bacterial spp. isolates	No. (%) of no growth cultures
Al- Hillah Teaching Hospital	512	13 (2.5%)	423 (82%)	76 (14%)
Medical City of Mirjan Hospital	588	4 (0.6%)	474 (80%)	110 (18%)
Total	1100	17 (1.5%)	897 (81%)	186 (17%)

Antibiotic Susceptibility was determined by disk-diffusion method, the Seventeen A. baumannii isolates exhibited different pattern of resistance to different antibiotic agents (Figures 1), we worked on selecting only carbapenem resistance Acinetobacter baumannii (CRAB) isolates that resist to carbapenem class (imipenem and meropenem) while discarded the susceptible isolates to this class. The number of CRAB isolates at the present study was 8 isolates which demonstrating highest resistance, also these isolates were resist to most antibiotic classes that tested (Figures 4-5 and 4-6), which include cephalosporin antibiotics (cefepime, ceftazidime and cefotaxime), penicillins (penicillin, carbenicillin, piperacillin and ampicillin-sulbactam), augmentin and aztreonam with percentage of resistance at (100%).

The resistance to other drug classes varied but still high among the isolates. The resistance for Aminoglycosides antibiotics (amikacin and gantamicin), were 3(37.5%) and 8(100%), respectively. The resistance to tetracycline was detected as 7(87.5%) isolates. For the following class of antibiotic include quinolones (ciprofloxacin and nalidixic Acid) and Fluorquinolones (norfloxacin and levofloxacin) displayed the high resistant rate 8(100%) isolates, but resistance to trimethoprim-Sulphamethoxazole was detected as 6(75%) isolates.

The resistance to polymyxins E (colstin sulphate) was displayed lowest resistance as 1(12.5%) isolate, whereas the resistance to phenicols (chloramphenicol) was detected as 6(75%) isolates. The results revealed that all tested isolates were resistant to at least 3 classes of antibiotics, hence these isolates were considered to be multidrug resistant (MDR).

Genotypic detection of NDM genes:

AmpC genes were appeared in (87.5%) of CRAB isolates PCR products using specific primers gene. Moreover, the amplification of DNA of CRAB isolates with blaOXA-23 and blaOXA-235 primers, observed presence of (75%) of blaOXA-23 and (12.5%) of blaOXA-235 gene positive isolates (Fig.2, Fig.3 & Fig.4).

Figure (2): Detection of ampC gene product (amplified size 663 bp) using DNA template of Acinetobacter baumannii isolates by PCR (agarose gel electrophoresis, 1.5% agarose, 70 volt for 90 mins.). Lane (M), DNA Ladder (100 bp). Lanes (1, 2, 3, 5, 6, 7and 8) of A. baumannii isolates show positive results. Only lanes (4) show negative results.

Figure (3): Detection of OXA-23gene product (amplified size 1066 bp) using DNA template of Acinetobacter baumannii isolates by PCR (agarose gel electrophoresis, 1.5% agarose, 70 volt for 90 mins.). Lane (M), DNA Ladder (100 bp). Lanes (1, 2, 3, 6, 7 and 8) of A. baumannii isolates show positive results. Lanes (4 and 5) show negative results.

Figure (4): Detection of OXA-235 gene product (amplified size 750 bp) using DNA template of Acinetobacter baumannii isolates by PCR (agarose gel electrophoresis, 1.5% agarose, 70 volt for 90 mins.). Lane (M), DNA Ladder (100 bp). Only lanes (7) of A. baumannii isolates show positive results.

4. DISCUSSION:

The present results agree with local study by Alsehlawi and his colleagues⁸ who found that isolation rate of Acinetobacter in Al-Najaf city was (1.5%) and Gales and his colleagues⁹ have showed that the Acinetobacter caused 1.5% of bacterial infections in five geographical regions (Canada, the United States, Latin America, Europe, and the Asia-Pacific) during 1997-1999. while is relatively in agreement with a studies carried out in Al- Diwaniya city by Al-Garaawi¹⁰ and in Hillah city by Al-Harmoosh¹¹ they found that isolation rates of Acinetobacter were (3.7%) and (0.76%) respectively, but Khalili and his colleagues¹² showed that the Acinetobacter form (17%) of nosocomial infections caused by Gram negative bacteria and Sahar and AL-Yasseen¹³ who recorded that A. baumannii isolates were (18%) from clinical samples, these results got disagreement with the present study.

The results from figure (1) showed that CRAB isolates has a high resistance level for cephalosporins (cefepime, ceftazidime and cefotaxime) (100%). This result is quite agreement with a pervious study in Iraq by, Alsehlawi and his colleagues⁸ and AL-kadhmi¹⁴ reported that resistance rate of A. baumannii isolates to cefepime, ceftazidime and cefotaxime and were (100%). The resistance to cephalosporins is multifactorial, one of these is the overproductions of acinetobacter-derived cephalosporinase (ADC) type AmpC resistance genes which conferring high-level resistance to expanded-spectrum cephalosporins and monobactams, extended-spectrum β-lactam antibiotics¹⁵. Or due to ESBL production which confers ability to hydrolyze third-generation cephalosporins and aztreonam. And because of increasing numbers of bacterial strains express different types of β-lactamases including inducible and / or plasmid mediated AmpC type of enzyme may also increase the chance for resistance to cephalosporins ¹⁶.

The present study showed a high level of resistance to penicillin, carbenicillin, piperacillin, ampicillin-sulbactam, A similar local result was recorded by Alsehlawi and his colleagues¹¹ who noted that that all A. baumannii isolates were resistant to penicillin, carbenicillin (100%) for each one, but (50%) to ampicillin-sulbactam. .Another study in Baghdad found that 100% of A. baumannii clinical isolates were resistant to piperacillin¹⁷. High resistance to this class of antibiotics may be due to widespread use of these antibiotics in Iraqi hospitals. The susceptibility profile results from Figure (1) showed that the antibiotic combination such as amoxicillin/clavulinic acid did not show promising activities against these isolates despite it being a recommended antibiotic treatment for Acinetobacter infections. This result in agreement with a pervious study at Babylon by Al-Harmoosh¹¹ who found that 8 (80%) A. baumannii isolates were resistant to amoxicillin/clavulinic acid. The high resistance rates found in this study may be associated with the high frequency at which these antimicrobial drugs were used for both empirical and therapeutic treatments of hospitalized patients. This practice may have exerted selective pressure leading to the emergence of multidrug resistant strains which in turn may have stimulated the acquisition of genes encoding resistance mechanisms¹⁸. This study demonstrated that the resistance of CRAB isolates to aztreonam were 100% this is because the production of ESBLs, since it mediates resistance to broad spectrum cephalosporins and aztreonam ¹⁹.

Results of the present study showed that amikacin was more effective (37.5%) than other aminoglycosides, gentamicin (100%). This result was agreement with study in Babylon by Al-Harmoosh¹¹ who found that resistance against aminoglycosides were (50%) to amikacin, whereas gentamicin (70%). In another study in Najaf by Alsehlawi and his colleagues⁸ who found that resistance against aminoglycosides were (58.3%) to amikacin, whereas gentamicin (83.3%). High efficiency of amikacin may be due to AMEs (aminoglycoside-modifying enzymes) which consider the source of this resistance. The genes encoding AMEs can be disseminated via integrons, and expression of AMEs enable bacteria to catalyze the modification of amino and hydroxyl groups on sugar moieties, such as aminoglycosides. This ability is a major cause of aminoglycoside resistance to clinical A. baumannii isolates²⁰. For tetracycline in this study the result of resistance was high (87.5%). This result was parallel with previous studies in Iran by Maleki²¹. This resistance is through a variety of mechanisms, including efflux pumps, ribosomal protection proteins, chemical molecule modification and target site modifications. Among Gram-negative organisms, the main mechanism leading to tetracycline resistance is through efflux pumps. The differences in gene-encoding efflux proteins account for the differences in resistance phenotypes to the different agents within the tetracycline class²².

As shown in figure (1) resistance to quinolones and fluroquinolone was (100%) which resistance is typically encoded chromosomally .In this study, resistance against fluroquinolones may reflect antibiotic pressure in Babylon hospitals. In China, Zhou and his colleagues²³ reported high resistance to quinolones (> 95%) among clinical isolates of A. baumannii. Alsehlawi and his colleagues⁸ who found that resistance against ciprofloxacin was (91.6 %). The outer membrane of A. baumannii has been associated with intrinsic antimicrobial resistance resulting from the over-expression of the adeABC and adeFGH efflux pump genes. These findings indicate that active efflux is involved in both intrinsic and acquired resistance to fluoroquinolones²⁴. Result of resistance to trimethoprim-sulfamethoxazole was (87.5%) it came in parallel with study in Turkey by Kulah and his colleagues²⁵. This may be due to multiresistance plasmid harboring A. baumannii ²⁶. The present study showed low resistant level to polymyxin E (12.5%). This result had been convergent with study at by Livermore and his colleagues²⁷ in London 2010, carbapenem-resistant and MDR Acinetobacter baumanii-infected and colonized patients were evaluated, and only colistin was found to have high in vitro efficiency against the isolated strains (99.4%). Colstin targets lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacteria. In there, colistin interacts with the lipid A component of the LPS, displacing the calcium and magnesium bridges that stabilize the LPS, leading to permeabilizing the bacterial outer membrane after that colistin inserted through these cracks in the outer membrane of bacteria causes ‘self-promoted uptake’ as well as disruption of the integrity of the inner membrane also that leads to bacterial killing. The majority of mechanisms of resistance to colistin are based on modifications to the lipid A portion of LPS of gram-negative bacteria, that reduces its net negative charge resulting in less electrostatic interaction with positively charged colistin molecule ²⁸. Finally, resisance to chloramphenicol was high, this result is quite compatible with study achieved by Sood²⁹ in India. This resistance came from enzymatic inactivation by acetylation mainly via acetyltransferases or, in some cases, by chloramphenicol phosphotransferases. Resistance to chloramphenicol may also be due to target site mutation/modification, decreased outer membrane permeability, and the presence of efflux pumps that often act as multidrug extrusion transporters, thereby reducing the effective intracellular drug concentration ^30.The AmpC β-lactamases (AmpC enzymes) are produced by some bacteria and their production is mediated either by chromosomes or by plasmids of Gram-negative bacteria. As a ‘serine’ cephalosporinase, AmpC β-lactamases cannot be inhibited by clavulanic acid, but can be inhibited by cloxacillin. The A. baumannii bacterium is equipped with the chromosome encoded enzyme of class C, and ampC genes from heterogeneous A. baumannii strains highly correlate with each other, but differ from those from other types of strains. Thus, these enzymes are termed as the acinetobacter-derived cephalosporinase ADC family^31.

In the current study found that 7(87.5%) of CRAB isolates was harbored AmpC genes (Table 1). In other local study at Al-Diwaniya city by Al-Khazaali³² who found AmpC genes in 13(81.3%) of A. baumannii isolates and another study conducted in India by Kaur and his colleagues³³ who found (13.3%) of A. baumannii isolates was harboring for AmpC genes. This is the first report on prevalence of AmpC genes in Babylon hospitals among A. baumannii isolates. Moreover, detection of AmpC is considered as a first report on prevalence of this gene among CRAB isolates in Iraq.

In this study, eight of A. baumannii were found to be resistant to carbapenem drugs and also highly resistant to cephalosporin and penicillins antibiotic classes except (37.5%) of isolates show intermediate resistance to ampicillin-Sulbactam. The results of the drug sensitivity test revealed a multidrug resistance pattern of AmpC A. baumannii. Carbapenem is the priority drug used for the treatment of infections with AmpC CRAB. Some lines of evidence indicate that sulbactam can irreversibly bind Acinetobacter PBP, which contributes to its inherent antimicrobial activity³⁴. However, this study demonstrated a drug resistance rate of approximately (62.5%) to ampicillin-salbactam but with intermediate level, complicating its drug-resistant mechanisms. We identify a low level of drug-resistant strains for colsin sulfate, which can be a priority drug for treatment. It has been reported that aztreonam in combination with polymyxin may improve the therapeutic effect ³⁴. Nonetheless, a kidney function test is necessary before the combined application of these two drugs, given that polymyxin may induce ototoxicity and renal toxicity. It has also been suggested that antibacterial peptides and vaccination may also be potential choices for therapeutic strategies³⁵.

The OXA β-lactamases were among the earliest β-lactamases detected; however, these molecular class D β-lactamases were originally relatively rare and always plasmid mediated. They had a substrate profile limited to the penicillins, but some became able to confer resistance to cephalosporins. From the 1980s onwards, isolates of Acinetobacter baumannii that were resistant to the carbapenems emerged, manifested by plasmid-encoded β-lactamases (OXA-23, OXA-40, and OXA-58) categorized as OXA enzymes because of their sequence similarity to earlier OXA β-lactamases. It was found that every A. baumannii strain possessed a chromosomally encoded OXA β-lactamase (OXA-51-like), some of which could confer resistance to carbapenems when the genetic environment around the gene promoted its expression. Similarly, Acinetobacter species closely related to A. baumannii also possessed their own chromosomally encoded OXA β-lactamases; some could be transferred to A. baumannii, and they formed the basis of transferable carbapenem resistance in this species. In some cases, the carbapenem-resistant OXA β-lactamases (OXA-48) have migrated into the Enterobacteriaceae and are becoming a significant cause of carbapenem resistance. The emergence of OXA enzymes that can confer resistance to carbapenems, particularly in A. baumannii, has transformed these β-lactamases from a minor hindrance into a major problem set to demote the clinical efficacy of the carbapenems³⁶.

Result from present study show increasing in prevalence of blaOXA-23 gene in contrast with previous local study. It was found 6(75%) of CRAB isolates carried blaOXA-23, while Al-Harmoosh¹¹ and Alsehlawi and his colleagues⁸ who found 4(40%) and 2(40%) of A. baumannii isolates had blaOXA-23 genes positive, respectively. The OXA-23 consider first group of carbapenem-resistant OXA-type lactamases identified in A. baumannii isolates. It was first identified in an A. baumannii isolate collected in Edinburgh, United Kingdom, in 1985, interestingly the same year that imipenem was first approved for use³⁷. The production of OXA-23 by an A. baumannii strain is enough to confer resistance to the carbapenems. When a low-copy-number plasmid vector carrying a cloned blaOXA-23 gene is put into sensitive A. baumannii type strain³⁸. However, when the blaOXA-23 gene is carried in a strain that also expresses the AdeABC efflux pump, the resistance is increased. This indicates that, unlike some of the other OXA-type carbapenemases, strains do not require other resistance mechanisms to work in synergy with OXA-23 to be carbapenem resistant, although high levels of resistance are achieved only when there are other mechanisms present³⁶. This explain the presence of another resistance mechanisms with blaOXA-23 gene in CRAB isolates of present study.

Finally, one isolate had been found harboring a novel OXA enzyme (blaOXA-235). In contrast with the study of Perez-Grueso and his colleagues³⁸ who found (80%) of A. baumannii was carrying blaOXA-235. The OXA-235 hydrolyzed penicillins and carbapenems but did not show activity against expanded-spectrum cephalosporins, as observed with other CHDLs. Similar to the latter enzymes, OXA-235 shows high affinity for the carbapenems but with low rates of hydrolysis. Despite this weak hydrolysis, it is very likely that OXA-235 contributes significantly to imipenem and meropenem resistance³⁹.

5. CONCLUSION:

Our study has shown the spreading of carbapenem resistance Acinetobacter baumannii (CRAB) which harboring genes as Ampc, blaOXA-23 and blaOXA-235 thus causing different infections. Hence, it is suggested that, such isolates, which consequently poses an increased threat to hospitalized patients in Hillah hospitals and more importantly, avoiding misuse and overuse of antibiotics may reverse the undesired effects of multidrug resistant.

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