Prevalence of ESBLs in Acinetobacter baumannii isolated from intensive care unit (ICU) of Ghaem hospital, Mashhad, Iran

1Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 2Research Center for Food Hygiene and Safety, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 3Department of Parasitology and Mycology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 4Department of Microbiology and Immunology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran. 5Department of Community Medicine, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 6Nursing Department Basic Sciences Faculty, Hamedan Branch, Islamic Azad University, Hamedan, Iran.

a genus of gram-negative bacteria belonging to the gammaproteobacterial 1 . Acinetobacter are rodeshape during rapid growth and coco-bacillary in the stationary phase. They are generally encapsulated, nonmotile, aerobic, gram-negative organisms with tendency to retain crystal violet and therefore to be incorrectly identified as gram-positive cocci 2 .
Frequent misidentification of Acinetobacter as Neisseria or Moraxella on gram staining is readily clarified by the negative oxidase reaction of Acinetobacter. Additionally, Acinetobacter are catalase-positive. Hemolysis of red blood cells, acidification of glucose, growth at 44°C, and variability in carbon source uptake are few of the phenotypic characteristics applied to distinguish Acinetobacter strains 3 , A. baumannii isolates are more likely caused disease in patients with immunosuppression, serious underlying disease and people who are exposed to invasive procedures accompanying treatment with broad-spectrum antibiotics. Therefore, the spread of these species in ICU and burn wards is more. A. baumannii is an important cause of nosocomial infection, such as ventilator associated pneumonia(VAP), urinary tract infections, wound infections, and septicemia 4

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A. baumannii is a significant opportunistic pathogen that mainly infects critically ill patients in ICU 5 .
As known the ability of A. baumannii to achieve different mechanisms of resistance, also, resistance to all available common antibiotics as well as lack of new effective antimicrobial drugs are the most important causes of risk about this organism. A. baumannii isolates which are resistant to three or more classes of antibiotics are called multi-drug resistant strains (MDR). Increasing antibiotic resistance in Acinetobacter inhibits from appropriate management in antibiotic therapy 6

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A. baumannii has several innate resistance mechanisms to a number of antibiotics, such as aminopenicillins, first-and second-generation cephalosporins and chloramphenicol. Besides this, it has a considerable capacity to acquire mechanisms conferring resistance to broadspectrum b-lactams, carbapenems, aminoglycosides and fluoroquinolones. Beta lactam antibiotics (mainly carbapenems) are now the first drug of choice to treat this microorganism; however, in the last decade, resistance to carbapenems has appeared in hospitals worldwide owing to the production of beta-lactamase, change in permeability, increase in efflux, and modification of the affinity of penicillin-binding proteins (PBP S ) in these bacteria 7 . Production of plasmid-mediated extended-spectrum b-lactamases (ESBLs) is one of the most important mechanisms of resistance against beta-lactam antibiotics. Many of these enzymes have evolved from TEM and SHV -lactamases, but recently a large number of ESBLs are related to TEM and SHV, such as GES and VEB, have been described 8 . Plasmid is accounted for distribution of the most beta lactamases; however, the gene encoding these enzymes may also be on the chromosome or transposable elements and integrons.
ESBLs are also able to hydrolyze three and four generation cephalosporins and monobactams. ESBLs producer isolates are inhibited by b-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam), At present, there are more than 300 different ESBL variants, and these have been clustered into nine different structures and these evolutionary families based on amino acid sequence 9 . TEM, CTX, SHV, GES, VEB, OXA-10 and PER are the major types.
In according to the information on the prevalence of these enzymes and antibacterial resistance pattern, control, prevention and treatment of this bacterium is important, thus, this study aimed to evaluate the prevalence of ESBLs in A. baumannii isolated from ICU of Ghaem hospital, Mashhad, Iran.

Bacterial sources
A total of 140 A. baumannii isolates were recovered from hospitalized patients in ICU of Ghaem hospital in Mashhad city from December 2014 to March 2015. All nonlactose fermenting members were subjected to microbiologic and biochemical tests such as; gram staining, oxidase, catalase, O/F, and growth at 42 °C on nutrient agar medium. For confirmation of A. baumannii isolates, API20NE kit (version 6.0, bio-Merieux, Marcy L'Etoile, France) was applied. Then until use, clinical isolates were stored in nutrient broth containing 20% glycerol at -80 °C.

Phenotypic Detection of Beta-Lactamase
In phenotypic confirmation of ESBLs producers on Muller Hinton agar, the combination disc test (CDT) was applied as previously defined 11 . Cefotaxime (30 µ) or ceftazidime disks (30 µ) with or without clavulanate (10 µ) were used. After incubation of plates for 24 h at 37 °C, if the diameter of inhibition zone for each of these antibiotics in combination with clavulanic acid compared to antibiotics alone, increased by more than 5 mm, they defined as the ESBL-producing isolates, if no, isolates were reported as ESBL negative. P. aeruginosa ATCC 27853 was applied for quality control of isolates.

DNA extraction and PCR
For DNA extraction and template preparation, the boiling method was used as previously described 12 and lastly samples stored at -20 °C, till use. The primer pair sequences designed by primer premier software for detection of ESBL genes in clinical isolates of A. baumannii using PCR technique are shown in Table 1. Of note, PCR of bla OXA-51 -like gene was also used for confirmation of isolates identification. The PCR program for bla GES , bla CTX and bla PER genes was composed of an initial denaturation step (94 °C, 5 min) followed by 30 cycles of denaturation step (94 °C, 1 min), annealing step (60 °C, 1 min), and extension step (72 °C, 1 min) with final extension (72 °C, 7 min). The DNA amplification program for bla oxa-10 , bla SHV , bla VEB and bla TEM genes was similar to previous genes except that the annealing temperature was 51 °C. Components of PCR master mix (Amplicon, Denmark) were as follows; 1.5 mM Mgcl 2 , 10 pmol/µl of each primer, 0.2 mM dNTPs, 1U Taq DNA polymerase, 1X PCR buffer and 50 ng/µl DNA. PCR products were analyzed using 2% agarose gel electrophoresis (Cinaagen, IRAN). And 50bp DNA ladder (Fermentas company product) was used to detect the specific PCR products related to the bla genes. Then, results were observed under UV light gel documentation system.

Sequencing of PCR products
The PCR products of three samples for each mentioned gene were subjected to direct sequencing and the nucleotide sequences were evaluated and analyzed with CLUSTAL W2 and BLAST softwares.

Data analysis
SPSS software (version 22, Chicago, IL, USA) was used for performing the statistical analy sis using chi-squire and Fisher's exact tests. Also, P-value<0.05 was con sidered as significant statistically.

RESULTS
In this study, a total of 140 isolates A. baumannii collected from ICU of Ghaem hospital in Mashhad, Iran, from December 2014 to March  were in the age group 31-50 years. Also, the most rates of isolates (with prevalence of 51.4%) were seen in female than male. As shown in Table 2, results of antibacterial susceptibility pattern revealed that in A. baumannii the high resistance was to all antibiotics except colistin, as resistance rates to imipenem, meropenem, ceftazidime, cefotaxime, cefuroxime, ceftriaxone, Cefepime, ertapenem and ampicillin/sulbactam were 97.9%, 98.1%, 96.4%, 97.9%, 99.3%, 97.9%, 97.9%,98.6% and 97.1%, respectively. The most effective antibiotic against A. baumannii was colistin with susceptibility 97.9% followed by amikacin with sensitivity 27.1 % ( Table 2).
It was also presented that none of the isolates were ESBL producers by Combination disk method. Although A. baumannii isolates exhibited a high degree of resistance to third-generation cephalosporins but they did not produce ESBL.
A. baumannii isolates which at one time had two ESBL genes were: bla PER /bla GES 2(1.4%),   In addition, results showed that there no correlation was found between prevalence of ESBLs genes and types of clinical samples (p>0.05), as per Table 4.

DISCUSSION
A. baumannii is mostly found in hospital settings and is nowadays noticed more than ever due to its survival ability in such environments and causing nosocomial infections. In this study, among the aminoglycosides, amikacin was resistant in 67.1% of cases and in other groups; cephalosporins, carbapenems, and penicillin were almost 100% resistant, indicating multiple drug resistance in these isolates. Colistin was the most effective antibiotic (97.9%). Our results demonstrated that the rate of resistance was significantly lower in colistin compared to other antibiotics. The possibly reason for low resistance to this antibiotic may be owing to its infrequent prescription during the recent period. In a study conducted by Shahcheraghi et al (2009) 14 , confirmed that most isolates were resistant to ceftazidime and cefepime. Regarding the present results and similar studies, due to over-administration of the third generation cephalosporins and lack of observing the hygienic principles in the community, a considerable resistance has been developed against this generation of cephalosporins. So, based these findings, the third generation cephalosporins are not good choice for treatment of infections caused by MDR A. baumannii isolates.
The study conducted by Srinivasan and colleagues in Ohio, USA 15 , more than 80% of the isolates were resistant to a wide range of cephalosporins and 20% to imipenem, while in our study, resistance to imipenem was more than 90%, which this dissimilarity might be due to unnecessary overuse of antibiotics. An another study conducted by Akan et al in 2002, in Turkey on 277 A. baumannii isolates revealed that the resistance rate to imipenem and amikacin was 53.6% and 59.8%, respectively 16 , in contrast, this rate was much higher in our study and also in comparable above mentioned ones. All of studies stated here used disk diffusion agar method to evaluate antibiotic susceptibility similar to our study. Therefore, the difference in results could be attributed to diversity in types of isolates, variety in antibiotic disks used, and difference in geographical regions of the studies and policies of infection control 17 . Thus, the regional determination of antibiotic susceptibility of A. baumannii can act as a suitable guide for effective use of routine antibiotics. Since in this study and similar ones, the most A. baumannii isolates (50.7%) were obtained from pulmonary secretions, it appears that the respiratory tract is the most involved in infections caused by A. baumannii. So, disinfection and sterilization of equipment and respiratory devices like respiratory is one of the ways for prevention of infection dissemination. Based on the studies conducted in our country (Iran) and through study reported by Sharif et al in 2013, 51% of A. baumannii isolates had the wide-range beta-lactamase-producing phenotype 18 . Also, Owlia et al in 2012, in Tehran reported that 21% of A. baumannii isolates had the wide-range beta-lactamase-producing phenotype 19 21 . In present study, we used the combination disk method similar to the method used in the mentioned studies, there was no positive test regarding phenotype. One probable reason for lack of production of extended b-lactamase-producing phenotype in the present study, compared to other studies, may be increased expression of AmpC genes and also beta-lactamases and metallo beta-lactamase enzymes. It is also possible that mechanisms other than extended b-lactamase like secretary pumps and variations in porins induce resistance in this organism. Indeed, resistance in A. baumannii is associated with a combination of various mechanisms including acquisition of b-lactamases, stable induction of AmpC, reduced permeability, changes in penicillin binding proteins, and somewhat, with an increase in Efflux pumps 21 . Performing of phenotypic tests alone is not able to determine the ESBL-producing isolates in A. baumannii. Some molecular tests need be performed to determine the presence of ESBL enzymes.
The bla OXA-51 gene is considered as a chromosol component of A. baumannii isolates which can used to identify this organism, 22 for this reason in present study we used bla OXA-51 gene for confirmation of the A. baumannii isolates.
Azhari and et al in 2010 in Tabriz indicated that among 100 isolates of A. baumannii from different clinical samples, PER gene was not found in any of samples 23 . The first report of presence of PER gene was detected in a study conducted by Farajnia and et al in 2013 in Tabriz, wherein its prevalence was 51.7% 24 which was higher than over results. But another in 2007 in Argentina presented that among 1 out of 6 isolates was positive for PER gene 25 31 . But in the study conducted by Ramoul and colleagues in 2013, CTX was not found in any of the isolates 32 , which is close to our results.
Several studies suggested varying distribution of resistant genes in different geographical regions. Geographical distance and also pattern of antibiotic usage can predispose to emergence of resistant genes in different geographical areas. As Beta-lactamase-producing isolates constitute a lower percentage compared to Beta-lactam-resistant isolates, it seems that in addition to the production of Beta-lactamases, other factors like the presence of Efflux pumps and cellular wall canals or purines also contribute to the creation of resistance. Due to the capacity of these isolates for transmitting resistance genes to other clinical isolates, the exact identification of Betalactamases genes contributing resistance is of most importance for care, treatment, and epidemiologic studies on transmission methods in hospitals 33 .
The high resistance of isolates to third and fourth generation of cephalosporins compared to the low number of ESBL producing isolates, proposed another resistance mechanisms such as secretory pump, purines, biofilm information involved in development of resistance.
Hence, the development in policies of antibiotic prescription and infection control are more critical to pre vent the spreading of such resistant infectious organisms.

CONCLUSION
According to the results, the high resistance was seen against selected antibiotics and the phenotypic tests are not sufficient alone for determination of ESBLs producer of A. baumannii isolates. So, molecular tests are also necessary for detection of these enzymes.