1. Introduction

The genus Acinetobacter is a member of the family Moraxellaceae in the order Pseudomonadales. More than 25 species within the genus Acinetobacter have been described ^[2]. The most important species of this genus is Acinetobacter baumannii which causes 2-10% of all Gram-negative infections in the Unites State and Europe ^[3]. It possesses little risk to healthy individuals, but generally causes infections in those with weakened immune systems specifically, the intensive care unit (ICU). The latter equipped with ventilators and invasive tools such as catheters that are factors that predispose to A. baumannii infections such as Ventilator Associated Pneumonia (VAP), meningitis, wound infection, septicemia, and urinary tract infections ^[3]. The clinical impact of Acinetobacter infection in terms of morbidity and mortality has been discussed widely in which the mortality rates range from 19% to 54% ^[4].

The infections caused by A. baumannii are often treated with cephalosporins including ceftazidime and ceftriaxone, aminoglycosides such as tobramycin and amikacin, carbapenems, and tetracycline. However, to date, most strains of A. baumannii have become increasingly resistant to all these currently available antibacterial agents ^[5]. The clinical significance of A. baumannii has grown significantly over the last few decades mainly due to the fact that this species possesses a variety of antibiotic resistance genes on plasmids, transposons and integrons and innate antimicrobial resistance mechanisms such as cell surface structures that prevent the influx of antibiotics which lead to failure of treatment ^[6].

Due to growing the numbers of A. baumannii infections and lack of new forms of antibiotics to treat the infections, some studies are focused to assess the in vitro combination activity of different types of currently used antibiotics against carbapenem-resistant A. baumannii such ascarbapenem/ sulbactam combination and colistin/ rifampicin combination. Some reports showed that the in vitro combinations of antibiotics such as imipenem/ sulbactam and colistin/tigecycline, proved effective against carbapenem resistant A. baumannii ^[7].

A. baumannii has already been compared to methicillin-resistant Staphylococcus aureus (MRSA), and has even been termed the ‘Gram-negative MRSA ^[8]. Also it has been identified as an ESKAPE pathogen (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species); a group of pathogens with a high rate of antibiotic resistance that are responsible for the a majority of nosocomial infections ^[9]. Colloquially, A. baumannii is referred to as 'Iraqibacter' due to its seemingly sudden emergence in military treatment facilities during the Iraq War ^[10].

In the literature, various terms have been used to describe the resistance rate of A. baumannii to antibiotics like Multidrug Resistant A. baumannii (MDR-AB) is used to describe the isolates which are resistant to at least three classes of antibiotics including Penicillins, cephalosporins, fluroquinolones and aminoglycosides. While the term Extreme Drug Resistant (XDR) is used when the isolates are resistant to the three above mentioned families plus carbapenems, Finally the Pandrug- Resistant (PDR) which is used to describe the A. baumannii which are (XDR) with resistance to polymyxins ^[11].

Susceptibility of A. baumannii to antimicrobials is considerably different among countries, among centers and even among the wards of a given hospital. These differences may reflect different patterns of antimicrobial usage and different epidemiological situations, including antimicrobial control measures and policies ^[12].

Regarding Sudan, there are no published records about the prevalence of A. baumannii in hospitals. However, some reports are available from other countries. In the Islamic Republic of Iran, for example, a prevalence of 15% was reported ^[13], in India it was 9.5% ^[14], and in Kuwait it was 22.1% ^[15]. In one study carried out in Saudi Arabia, A. baumannii was the most common isolated organism among Gram-negative bacteria, with a prevalence of 31.7% ^[16].The variations in the prevalence and resistance patterns among isolates stress the importance of local surveillance to determine the best antimicrobial therapy for A. baumannii infections.

This study aimed to determine the prevalence and antibiotic resistance pattern of A. baumannii isolated from various clinical specimens at Royal Care International Hospital (RCIH) Khartoum, Sudan.

2. Materials and Methods

This retrospective study was carried out over a period of 37 month from July 2011 to August 2014 in the department of microbiology, (RCIH) located in Khartoum, Sudan. Consecutive, non-duplicate (275) isolates of A. baumannii were recovered from various clinical specimens, namely; Sputum, wound swabs, urine, blood, soft tissue, Central Venous Catheter Tip (CVC) and other body fluids (CSF, synovial and ascitic fluid).

The samples were collected and processed during the course of routine diagnostic work up from patients in the ICU, wards and outpatient departments of the hospital. The specimens received in the laboratory were inoculated on 5% Blood Agar and MacConkey Agar and incubated overnight aerobically at 37°C. Blood specimens were inoculated on tryptone soya broth (Hi-Media, Mumbai) and then sub cultured on chocolate agar and MacConkey agar. A. baumannii isolates were initially identified by colonial morphology, Gram staining, growth at 37°C, a negative oxidase test, and oxidation of glucose. API E20 (BioMe´rieux, Marcy l’Etoile, France) were used to confirm the identification of the isolates ^[17]. Antimicrobial susceptibility was done by disc diffusion method as per the (CLSI) guidelines ^[1], using Muller-Hinton agar (Hi-Media, Mumbai) and antimicrobial discs (bioanalyse, Turkey and Hi-Media, Mumbai). The following antimicrobial agents (μg) were used: Ceftazidim (30), cefepime (30), cefuroxime (30), gentamicin (10), amikacin (30), ciprofloxacin (5), amoxiclav (30), meropenem (10), cephalexin (30), ceftriaxone (30) aztreonam (30) and colistin (10). The diameter of inhibition zones was measured and data were reported as susceptible and resistant. Quality control of the disks was checked by using reference strains.

3. Results

During the study period the total number of pathogenic Gram negative isolates, was 2899, of which 275 isolates were A. baumannii (9.5%). The majority of A. baumannii was isolated from sputum 61% followed by wound 19%, blood 9%, urine 3%, and others including: pus, CSF, synovial fluid, bone, soft tissue, CVC tip, ascitic fluid was 8% (Table 1).

Of the 275 A. baumannii isolates, 198 were isolated from ICU patients (72%), while 65 (24%) were from inpatients and 12 (4%) from outpatients department (Figure 1).

In this study most of A. baumannii isolates were highly resistant to the tested antibiotics, 92% were resistant to cefepime, 96% to ceftazidime, 99% to ceftriaxone, 100% to cefuroxime, 100% cephalexin, 92% gentamicin, 81% amikacin, 91% ciprofloxacin, 98% amoxiclav, 89% meropenem, 95% aztreonamand, 37% colistin (Figure 2).

Table 1. The Numbers and prevalence of A. baumannii clinical isolates from various clinical samples

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Figure 1. prevalence of the A. baumannii among ICU, In patient (IN) and out patients (OUT) at RCIH

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Figure 2. Resistance pattern of A. baumannii isolates against different antibacterial agent

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4. Discussion

In this study, the prevalence of A. baumannii in RCIH, Sudan was 9.5% which is identical or similar to those from developing countries like India where the rate were 9.5% ^[14], 9.4% ^[18] and 11% ^[19] respectively. In Japan the prevalence rate was 18% ^[20], in Kuwait 22.1 ^[15] and in Saudia Arabia 31.7 ^[16] which are higher than that in the current study.

Among the source of the isolates, most of A. baumannii was isolated from sputum specimen (61%).Other studies similar to this, were carried out by Jaggi et al., ^[19] where the maximum isolates were obtained from sputum 54.6% and Villerset al., ^[21] reported a predominance of A. baumannii in tracheo-bronchial secretions 48.8%. All these findings may support the presence of A. baumannii as a common colonizer in the respiratory tract, Its isolation from sputum without clinical signs and symptoms and may not necessary mean that they are causing infection. They can be only colonizer ^[22].

In this study the A. baumannii was mostly isolated from ICU patents (72%) followed by inpatient (24%) and outpatient (4%).This is in agreement with most of the global studies that reported the predominance of A. baumannii isolated from ICU patients, ^{[18, 23, 24, 25]} They concluded that A. baumannii is the ICU Superbug. These results collaborates the truth that a lot of risk factors connected with A. baumannii infection exist in the ICU like potential environmental reservoirs for A. baumannii, opportunities for cross transmission, immunocompromised patients who are colonized, patients having multiple wounds and indwelling devices and frequent contamination of the hands of health care workers responsible for patient care.

In the community, A. baumannii has been found to be associated with community acquired pneumonia (in patients with COPD, renal failure, diabetes mellitus, heavy smokers or excessive alcohol consumers or bacteremia in Asia and Australia, although rare in USA as evidenced by previous studies ^[26]. This supports the isolation of A. baumannii from the outpatient department in this study.

The resistance profile of the A. baumannii isolates at (RCIH) hospital showed that 97% were MDR which resistant to three groups of antibiotics include aminoglycosides, fluoroquinolones and cephalosporins. 89% were Extreme XDR. This is considered high resistance rate when compared to two other studies in one of which MDR were 82.4% of his isolates and XDR 65.0% ^[27] and in the other MDR were 72% and XDR 58% ^[28]. The differences observed between the studies can be due to the methods and resistance patterns which are influenced by environmental factors and antimicrobial susceptibility pattern used ^[29].

The emergence of PDR A. baumannii represents a major problem in health care settings because the colistin is the only therapeutic option available for treatment of infections caused by PDR

A. baumannii. Unfortunately, resistance to colistin was reported worldwide (Marchaim et al., 2011 ^[30]; Mammina et al., 2012 ^[31]; Lesho et al., 2013 ^[32]). In the present study, colistin resistance has been reported as 37% which is considered high when compared to other reports like the recent study from Malaysia carried out by Soo-Sum Lean et al., [33] in which the resistant rate was (25.9%). Therefore, rationale use of colistin is highly recommended whenever possible to reduce the emergence of resistance to such antibiotic.

In conclusion, we report for the first time in Sudan the prevalence and resistance rate of A. baumannii. This study is limited because it does not sharply identify patients with true infections versus those who are colonized but the present report is alarming because the emergence and increase in the rate of PDR A. baumannii which can be due to excessive antibiotic misuse in our region.

Hence we recommend: (1) Further molecular epidemiology study recommended to observe any MDR clone spreading in our region and to compare our MDR clone to any international clones that are spreading worldwide. (2) Judicious use of antibiotics by making an attempt to distinguish colonization from true infections and treatment should be only given to the clinically confirmed infections and not colonization. (3) Continuous monitoring and updating the antibiogram of bacteria in the hospitals. (4) Applying standardized infection control policies to minimize the spread of multidrug resistant bacterial infections.

References

[1]	Wayne, PA: Clinical and Laboratory Standards Institute; 2011. CLSI. Performance standards for antimicrobial susceptibility testing. 20th Informational Supplement. CLSI document M100-S21. Schreckenberger PC, Daneshvar MI, Weyant RS, Hollis DG. Acinetobacter, Achromobacter, Chryseobacterium, Moraxella, and other nonfermentative gram-negative rods. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Manual of Clinical Microbiology. Washington, DC: ASM Press, 2007; 8: 770-779.
	In article
[2]	Fournier, P. E., D. Vallenet, V. Barbe, S. Audic, H. Ogata, L. Poirelet al., Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLOS Genet. 2006; 10: 2-7.
	In article		CrossRef
[3]	Eveillard M, Soltner C, Kempf M, Saint-Andre J P, Lemarie C, Randrianarivelo C. et al,.The virulence variability of different Acinetobacter baumannii strains in experimental pneumonia. J Infect. 2010; 60 (2): 154-61.
	In article		CrossRef PubMed
[4]	Howard A, O'Donoghue M, Feeney A, Sleator RD. Acinetobacter baumannii: an emerging opportunistic pathogen. Virulence, 2012; 1: 3 (3): 243-50.
	In article
[5]	Kraniotaki E, Manganelli R, Platsouka E, Grossato A, Paniara O, Palù G. Molecular investigation of an outbreak of multidrug-resistant Acinetobacter baumannii, with char-acterisation of class 1 integrons. Int J Antimicrob Agents. 2006; 28: 193-9.
	In article		CrossRef PubMed
[6]	Song JY, Kee SY, Hwang IS, Seo YB, Jeong HW, Kim WJ. et al., In vitro activities of carbapenem/sulbactam combination, colistin, colistin/rifampicin combination and tigecycline against carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother. 2007; 60 (2): 317-22.
	In article		CrossRef PubMed
[7]	Rello J. Acinetobacter baumannii infections in the ICU: customization is the key. Chest1999; 115: 1226-1229.
	In article		CrossRef PubMed
[8]	Rice, LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis. 2008; 197 (8): 1079-81.
	In article		CrossRef PubMed
[9]	Drummond, Katie. Pentagon to Troop-Killing Superbugs: Resistance Is Futile. Wired.com. Condé Nast. Retrieved 8 April 2013.
	In article
[10]	Manchanda V, Sanchaita S, and Singh NP. Multidrug Resistant Acinetobacter. J Glob Infect Dis.: 2010; 2 (3): 291-304.
	In article		CrossRef PubMed
[11]	Cisneros JM, Rodriguez-Bano J. Nosocomial bacteremia due to Acinetobacter baumannii: epidemiology, clinical features and treatment. Clin. Microbiol. Infect: 2002; 8: 687-69.
	In article		CrossRef PubMed
[12]	Shakibaie MR, Adeli S, Salehi MH. Antibiotic resistance patterns and extended-spectrum β-lactamase production among Acinetobacter spp. isolated from an intensive care Unit of a hospital in Kerman, Iran. Antimicrob Resist Infect Control: 2012; 1: 1-8.
	In article		CrossRef PubMed
[13]	Patwardhan RB, Dhakephalkar PK, Niphadkar KB, Chopade BA.A study on nosocomial pathogens in ICU with special reference to multiresistant Acinetobacter baumannii harboring multiple plasmids. Indian J Med Res: 2008; 128: 178-187.
	In article		PubMed
[14]	AbdAllah S et al., Nosocomial infections and their risk factors at Mubarak Al-Kabeer hospital, Kuwait. Medical Journal of Cairo University, 2009, 78: 123-131.
	In article
[15]	Al Johani SM1, Akhter J, Balkhy H, El-Saed A, Younan M, Memish Z. Prevalence of antimicrobial resistance among gram-negative isolates in an adult intensive care unit at a tertiary care center in Saudi Arabia. Ann Saudi Med.: 2010; 30: 364-369.
	In article		PubMed
[16]	Forbes BA, Sahm DF, Weissfeld AS. Bloodstream infections. In: Wilson L, editor. Bailey and Scott's Diagnostic Microbiology, 12 th ed. St Louis: The Mosby Company; 2007; 778-97.
	In article
[17]	Jaggi, Namita; Sissodia, Pushpa; Sharma, Lalit, Acinetobacter baumannii isolates in a tertiary care hospital: J Microbiol Infect Dis: 2012; 2 (2) p 57.
	In article		CrossRef
[18]	H. Siau, KY Yuen, SSY Wong. The epidemiology of Acinetobacter infections in Hongkong, J Med Microbiol 1996; 44: 340-347.
	In article		CrossRef PubMed
[19]	Endo S., Yano H., Hirakata Y., Arai K., Kanamori H., Ogawa M.,et al. Molecular epidemiology of carbapenem-non-susceptible Acinetobacter baumannii in Japan. J. Antimicrob. Chemother: 2012; 67 (7): 1623-1626.
	In article		CrossRef PubMed
[20]	Villers D, Espaze E, Coste-Burel M, Giauffret F, Ninin E, Nicolas F. et al,. Nosocomial Acinetobacter baumannii infections: Microbiological and clinical epidemiology. Ann Intern Med 1998; 129: 182-189.
	In article		CrossRef PubMed
[21]	Dijkshoorn, L, Nemec, A, Seifert, H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol.: 2007; 12: 939-51.
	In article		CrossRef PubMed
[22]	Seifert H, Dolzani L, Bressan R, Van Der RT, van Strijen B, Stefanik D, Heersma H, et al,. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii. J ClinMicrobiol 2005; 43 (43) 28-35.
	In article		CrossRef
[23]	Wilks M, Wilson A, Warwick S, Price E, Kennedy D, Ely A, et al,. Control of an outbreak of multidrug-resistant Acinetobacter baumannii-calcoaceticus colonization and infection in an intensive care unit (ICU) without closing the ICU or placing patients in isolation. Inf Control HospEpidemiol, 2006; 27: 654-8.
	In article		CrossRef PubMed
[24]	Munoz-Price LS, Weinstein RA. Current concept: Acinetobacter Infection. N EnglJ Med 2008; 358: 1271-81.
	In article		CrossRef PubMed
[25]	Glow RH.,Moellering RC, Kunz LJ. Infections with Acinetobacter calcoaceticus (Herelleavaginicola): Medicine (Baltimore): 1977; 56: 79-97.
	In article
[26]	Evans BA, Hamoud, A, Towner S.A, Khan S.A, Amyes S.G. High prevalence of unrelated multidrug-resistant Acinetobacter baumannii isolates in Pakistani military hospitals. Int J Antimicrob Agents: 2011; 37: 580-581.
	In article		CrossRef PubMed
[27]	Dent L, Dana R M, Siddharth P. Multi-drug resistant Acinetobacter baumannii: a descriptive study in a city hospital. BMC Infectious Diseases: 2010; 10: 196-204.
	In article		CrossRef PubMed
[28]	Rit K, Saha R. Multidrug-resistant Acinetobacter infection and their susceptibility patterns in a tertiary care hospital. Niger Med J.: 2012; 53: 126-8.
	In article		CrossRef PubMed
[29]	Marchaim D, Chopra T, Pogue JM, Perez F, Hujer AM, Rudin S, et al,. Outbreak of colistin-resistant, carbapenem-resistant Klebsiella pneumoniae in metropolitan Detroit, Michigan. Antimicrob. Agents Chemother: 2011; 55 (2): 593-599.
	In article		CrossRef PubMed
[30]	Mammina C, Bonura C, Di Bernardo F, Aleo A, Fasciana T, Sodano C.et al,. Ongoing spread of colistin-resistant Klebsiella pneumoniae in different wards of an acute general hospital, Italy, Euro Surveill: 2011; 17 (33): 20-48.
	In article
[31]	Lesho E, Yoon EJ, McGann P, Snesrud E, Kwak Y, Milillo M, et al. Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections. J. Infect. Dis.: 2013; 208 (7): 1142-1151.
	In article		CrossRef PubMed
[32]	Lean SS, Suhaili Z, Ismail S, Rahman NI, Othman N, Abdullah FH, et al,. Prevalence and Genetic Characterization of Carbapenem-and Polymyxin-Resistant Acinetobacter baumannii Isolated from a Tertiary Hospital in Terengganu, Malaysia. ISRN Microbiology Vol 2014 (2014), Article ID 953417, 9.
	In article