Genetic background of carbapenem resistant Acinetobacter baumannii in a health care setting in Alexandria , Egypt

Acinetobacter baumannii (A. baumannii) is recognized as a major pathogen causing nosocomial infections, particularly in patients admitted to intensive care units (ICU), and many widespread strains are resistant to almost all antibiotics currently in use. Carbapenemases belonging to molecular class D (OXA enzymes) have emerged globally as the main mechanism responsible for this resistance. This work aimed at detecting the spread of OXA carbapenemases in A. baumannii in ICU patients and to identify the susceptibility patterns of strains to polymyxin E (colistin), polymyxin B, and tigecycline as a promising option for treatment. Twenty seven clinical isolates of A. baumannii, collected from Alexandria Main University Hospital, were included in this study. A. baumannii was isolated from different clinical samples from ICU patients. All 27 isolates were subjected to complete identification and antimicrobial susceptibility testing. The identified carbapenem resistant A. baumannii strains were tested for the presence of bla OXA-23, blaOXA-24, blaOXA58 and blaOXA-51 genes using multiplex PCR assay. Colistin and polymyxin B were found to be active upon the majority of identified resistant A. baumannii strains, where around 70% of the strains were sensitive to both of them, while tigecycline was found to be more effective as 92.5% of strains were sensitive to it. Resistance to carbapenems was mediated by both OXA-51 (100%) and OXA-23 (92.5%) for the tested A. baumannii strains.


INTRODUCTION
Acinetobacter baumannii (A. baumannii) is a Gram-negative organism that is increasingly recognized as a major pathogen causing nosocomial infections, including bacteremia and ventilator-associated pneumonia, particularly in patients admitted to intensive care units (ICU). (1)It is associated with multiple antibiotic resistance, and many widespread strains are resistant to almost all antibiotics currently in use, leaving few therapeutic options remaining. ( A. baumannii persistence in hospital environments and propensity to cause outbreaks are contributed to its increasing Bull High Inst Public Health Vol.41 No. 3 [2011]   resistance to antimicrobial drugs, including carbapenems, and resistance to desiccation and disinfectants.(3, 4) Several studies have shown the geographically widespread occurrence of multidrug-resistant A.
baumannii strains, which suggested a clonal relatedness of these strains.OXA-24-like; OXA-51-like; and OXA-58. ( Recently it has been suggested that enzymes belonging to the OXA-51-like subgroup are intrinsic to A. baumannii, occurring in most or all strains, although they are very variably expressed.A significant contribution to resistance by OXA-51-like enzymes therefore requires the presence of an insertion element ISAba1 upstream of the gene, able to act as a strong transcriptional promoter. ( The bla OXA-23 gene has been increasingly reported worldwide.For polymyxin E (colistin) and polymyxin B, the zone diameter were (resistant ≤12 mm, intermediate= 13, susceptible ≥14 mm).
Isolates were considered susceptible to tigecycline if they had an inhibition zone diameter of ≥13 mm. (18-21) A. baumannii strains were considered to be carbapenem resistant when they were found to be resistant to all beta-lactams, including carbapenems (carbapenemase producing bacteria).

Detection of genes coding for carbapenemases production
Multiplex PCR assay: All 27 identified resistant A. baumannii strains and carbapeneme sensitive A.
baumannii as a negative control isolated from the same ICU, were tested for the presence of bla OXA-23, blaOXA-24, blaOXA-58 and blaOXA-51 genes using multiplex PCR assay as follows: (22)

1) DNA Extraction
DNA was extracted from the 27 carbapenem resistant A. baumanii isolates by boiling five colonies of fresh organism culture in 20 μl of sterile distilled water for 5minutes, followed by centrifugation for 2 min at 8000 rpm, them supernatants were used for amplification.

RESULTS
In a 7 months period, a total of 27 A.
baumannii strains were isolated from different clinical samples collected from ICU patients admitted to Alexandria Main University Hospital.In this study, we examined A.
baumannii resistant strains that were found to be resistant to all beta-lactams, including carbapenems (carbapenemase producing bacteria).
They also observed that all their isolates were MDR and were totally resistant to imipenem, ampicillin/sulbactam, ciprofloxacin, and cephalosporins, with a high resistance rate to amikacin and trimethoprim/sulfmethoxazole (90% each), and gentamicin (85%).These results suggests the role of A. baumanii as a major pathogen causing VAP that requires more attention in infection control programs.
Another study in Alexandria, isolated A. baumannii were sensitive to colistin and 70.3% were sensitive to polymyxin B (Table 4).Mohammed and Raafat (28) demonstrated higher sensitivity results where colistin and polymyxin retained their activity in 82.6 % and 91.3% of the tested isolates, respectively.
High sensitivity to colistin were also reported, one study found that 98% of carbapenemresistant A. baumannii were susceptible to colistin.(25) In the present study it was found to be more effective against A. baumannii than the polymyxins.Around 92.5% of strains were sensitive to tigecycline.While tigecycline gives good in vitro activity against A. baumannii isolates, the question remains of its effectively in vivo. (37)On the contrary to our results, a study conducted in Egypt found only 60% of A. baumannii strains to be sensitive to tigecycline.In the present study, all the carbapenemresistant A. baumannii strains were tested for OXA-23, OXA-24, OXA-58 and OXA-51 genes using PCR assay.All strains (100%) were positive for bla OXA-51 gene, and all except 2 were positive for bla OXA-23 ( class D have emerged globally as the main mechanism responsible for this resistance.The OXA carbapenemases of Acinetobacter spp.are divided into four phylogenetic subgroups: OXA-23-like; PCR amplification for detection of OXA-23, OXA-24, OXA-58 and OXA-51 genes coding for class D carbapenemases were sought by PCR using Taq polymerase with specific primers supplied by Eurofins (Table 2).A 50-µl PCR reaction mixture containing 2 µl extracted DNA , 250 pmole of each primer, 25 µl of DreamTaq TM Green PCR Master mix( Fermantas).Amplification was performed in a Biocycler Tc-S Thermocycler (Boeco, Germany).The amplification profile started by 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 25 sec, annealing at 52°C for 40 sec, and extention at 72°C for 50 sec, followed by a final extension step of 6 min at 72°C.The amplification products were analyzed by agarose gel electrophoresis and ethidium bromide staining.

baumannii
which was found resistant to imipenem and 96% were MDR.The reason for this high level of resistance might be a nation wide problem due to the extensive use of carbapenems creating a selective antibiotic pressure and adding to the increasing trend of carbapenem resistant A. baumannii. (28)The increasing prevalence of resistant A. baumannii strains to all antibiotics, including carbapenems has led to the revival of interest in using polymyxins discovered more than 50 years ago, due to the limited choice of a suitable antibiotic drug of choice.(29, 30) Polymyxins in recent studies ls shown promise in the treatment of infections caused by MDR Gram-negative bacteria, including A. baumannii infections.In the present study, the susceptibility pattern for colistin, polymyxin B and tigecycline were tested.It was found that 74% of isolated A.
among 24 tested antibiotics was the only effective one against all MDR isolates.Tigecycline is a relatively new antibiotic approved by the FDA in 2005.Tigecycline is a minocycline active that has potency Bull High Inst Public Health Vol.41 No.3 [2011] against both Gram-positive and Gramnegative bacteria including Acinetobacter.

MATERIAL AND METHODS A. baumannii isolates:
(I) and resistant (R) according to standard tables published by Clinical Laboratory Standard Institute (CLSI).The inhibition zones for polymyxin E (colistin), polymyxin B zone and tigecycline were interoreted according to published recommendations.

Table 4 : Susceptibility patterns of the 27 A. baumannii strains to colistin, polymyxin B and tigecycline.
All A. baumannii strains had the bla OXA- 51 gene and with the exception of 2 strains they all also harbored bla OXA-23 .None of the tested strains was positive for bla OXA-58

Table 5 )
. The genes bla OXA-24 and bla OXA-58 were not detected in any of the isolates.