Multidrug-Resistant Acinetobacter baumannii: An Emerging Health Threat in Aseer Region, Kingdom of Saudi Arabia

Objective The study aims to determine the prevalence of multidrug-resistant A. baumannii in Aseer Region, Kingdom of Saudi Arabia. Methods This study evaluated the antibiotic susceptibility of ninety-four (n = 94) clinical isolates of A. baumannii. The isolates were collected from the south region of Saudi Arabia, and notably Aseer Region, during the period from 15 October 2014 to 15 January 2015. The isolates were tentatively identified as A. baumannii by routine bench tests and were confirmed by using VITEK® 2 Compact. The latest instrument was used to identify antibiotic susceptibility of these isolates. Results Antibiotic susceptibility in this study showed that 69% of these isolates were multidrug-resistant strains. Moreover, they were highly resistant to carbapenem drugs. Several strains of these isolates were found to be extremely resistant to test antibiotics and were only sensitive to one or two of them. Conclusion High rate of multidrug-resistant A. baumannii bacteraemia has emerged in the south region of Saudi Arabia as an important health problem. Therefore, it is considered as a new threat in hospitals, which requires a tremendous effort to stop its escalation and spread.


Introduction
Acinetobacter baumannii is a strictly aerobic, nonmotile, Gram-negative, nonfermentative, oxidase-negative, and catalasepositive organism. Within the healthcare settings, Acinetobacter baumannii has become a common pathogen, which can infect the respiratory tract, blood, soft tissues, and urinary tract of an individual. It is the etiological agent of nosocomial infections resulting in septicemia, meningitis, endocarditis, pneumonia, wound, and urinary tract infections [1,2].
ere are 32 Acinetobacter named and unnamed species, which have been identi ed [3].
e Acinetobacter species cause infections, which are associated with increased morbidity and mortality rates [4,5].
A. baumannii is tolerant to wide ranges of temperature, pH, and humidity. Studies have shown that this bacterium can survive on dry surfaces for 5 months, posing a challenge to hospital infection control measures [4]; therefore, this pathogen is considered as progressively important nosocomial pathogen, which can cause outbreak of serious infections. Despite the fact that the organism is often nosocomial, initial infection can be transmitted by patients, admitted from other hospitals [6,7].
Almost 25 years ago, A. baumannii was found to be resistant against antimicrobial drugs, such as aminopenicillins, cephalosporins, rst-and second-generation cephalosporins, cephamycins, aminoglycosides, ureidopenicillins, chloramphenicol, and tetracyclines. Strains of A. baumannii have started to acquire resistance to newly developed antimicrobial drugs and become prevalent in many hospitals [8]. More recently, the term "extensively drug-resistant" A. baumannii (XDRAB) has been used to characterize bacterial isolates resistant to all authorized antibiotics except two categories of antibiotic such as tigecycline and polymyxins [9]. e possibility of A. baumannii isolation from hospitalized patients is related to some important factors, such as bacterial colonization, medical sta -to-patient ratio, and other ward characteristics [10]. e current study was designed to determine the prevalence of multidrug-resistant A. baumannii in Aseer Region. Bacterial strains were isolated from patients who attended Aseer Central Hospital, Aseer Region, Kingdom of Saudi Arabia.

1. Ethical Approval.
is research was approved by Research Ethics Committee, College of Medicine, King Khalid University, Abha, Saudi Arabia.

Bacterial Isolates. 94 clinical isolates of A. baumannii
were collected from the south region of Saudi Arabia and notably Aseer Region (Figure 1) during the period from 15 October 2014 to 15 January 2015. All these strains were initially isolated and identi ed in Aseer Central Hospital laboratory and were sent to Department of Microbiology, College of Medicine, King Khalid University, for further assessment.

Media and Reagents.
Two culture media were used in this experiment: blood agar (Oxoid Ltd., Basingstoke, and Hampshire, United Kingdom) and MacConkey agar (Oxoid). MacConkey agar was used as a selective and di erential medium; while, blood agar was used for other processes, such as the sensitivity test. Biochemical reagents, such as catalase, oxidase, and indole, were used as initial identi cation tests.

Bacterial Identi cation and Puri cation.
All clinical isolates were cultured on blood and MacConkey agar and then tested for Gram staining to identify Gram reaction and bacterial morphology. In order to ensure that all isolates are pure, they were cultured several times. Several biochemical tests were undertaken to con rm that all these isolates were belonging to A. baumannii, such as catalase, oxidase, and indole tests. Identi cation of all strains was done, using the automated VITEK 2 system (bioMérieux) following manufacturer instructions as brie y described below.

Antibiotic Susceptibility of A.
baumannii. All isolates were tested for antibiotic susceptibility, using the automated VITEK 2 Compact system. All samples were cultured on blood agar, and then, a suspension was made for every isolate. Liquid suspension of all isolates was loaded on the VITEK system and left overnight to get the results. e next day, the results have illustrated the identi cation and antibiotic susceptibility of loaded samples.
VITEK 2 system was used for authenticating names of Acinetobacter species as described by the manufacturer (bioMérieux Inc., Durham, NC 27712, USA). e VITEK card contains 64 wells, which hold di erent uorescent biochemical assays. Out of the 64, 20 were carbohydrate assimilation; 4 were phosphatase, urea, nitrate, and actidione tests. e VITEK 2 machine controlled the card automatically including the lling, sealing, and then transferring the cards into the linked incubator (35°C). Each output report is decoded according to a particular algorithmic system. e acquired results were compared to the ID-GN (identi cation of Gram-negative bacteria) databank. In case of most known Acinetobacter species with a clear cut pro le, the system led to a correct identi cation of the unknown organism.

Identi cation of Tested
Isolates. Colony characteristics of isolated colonies were obtained using blood and MacConkey agar. On MacConkey agar, colonies of A. baumannii appeared as a nonlactose fermenter. On blood agar, colonies of all isolates were appeared as nonpigmented, domed, and mucoid, with smooth-to-pitted surfaces in a diameter of 1-2 mm colonies. All strains of A. baumannii were found to be catalase positive and oxidase/indole negative. e isolates were tentatively identi ed as A. baumannii by routine bench tests and have been con rmed by using VITEK 2 Compact.

Antibiotic Susceptibility of A. baumannii.
Antibiotic susceptibility testing has shown that 69% of these isolates were multidrug-resistant strains (Figure 1). 34 isolates were resistant to all antibiotics of interest and were only sensitive to colistin. e results have also shown that 15 of these isolates were only sensitive to colistin and a combination of trimethoprim and sulfamethoxazole, whereas 21 of isolates were sensitive to the combination of trimethoprim and sulfamethoxazole. One isolate and other two of them were resistant to all tested antibiotics except piperacillin and amikacin and tobramycin, respectively. e correlation between the number of isolates and their sensitivity to all tested antibiotics is shown in Figure 2.

Discussion
Multidrug-resistant A. baumannii is currently a major cause of respiratory infections in intensive care unit patients. However, Acinetobacter was identi ed as a low-virulent pathogen, which infects mostly immunocompromised hosts; however, the emergence of resistant strains to antibiotics over the past several years has increased. erefore, the concern toward this phenomenon has increased as well. MDR A. baumannii is recognized as one of the most di cult nosocomial pathogens to be treated and controlled [11,12].
In this study, multidrug-resistant and extensively resistant A. baumannii strains have been isolated and identi ed in the southern region of Saudi Arabia. e study has shown that 74% of these isolates are multidrug-resistant strains. Among these MDR strains, approximately 50% are extensively drugresistant isolates, which are sensitive to colistin and resistant to all other drugs of choice. is should raise the concern toward this potentially dangerous pathogen. e most effective antibiotic with high spectrum to treat A. baumannii is colistin (60%) and then the combination of trimethoprim and sulfamethoxazole (46%); these rates of spectrums were approximately compatible with previous studies [13].
A. baumannii isolates were extremely resistant to two carbapenem drugs: imipenem and meropenem. Among tested strains, only 5 (0.05%) and 4 (0.04%) isolates were susceptible to imipenem and meropenem, respectively. is process indicated that these isolates were more dangerous than previously identi ed MDR strains, which showed more than 90% susceptibility to both drugs [8].
Susceptibility of A. baumannii to carbapenems showed a clear drop, and it denotes a main epidemiological worry. e resistance mechanism was found due to class D OXAtype enzymes (oxa-23 and oxa-24/40) with carbapenemase activity [14]. is fact made the medical choices very limited mainly based on polymyxin combinations with other antibiotics. Carbapenem-resistant Acinetobacter spp. have Canadian Journal of Infectious Diseases and Medical Microbiology been described from many parts of the world, and Saudi Arabia is not an exception [15]. ese authors examined 64 Acinetobacter baumannii isolates during 2013 and 2014 from four di erent medical centers in Saudi Arabia and Egypt. All the isolates were found to be resistant to ceftazidime and cipro oxacin. ey reported the emergence of ST236 in Saudi Arabia and Egypt and the spread of distinct carbapenemresistant A. baumannii clones belonging to ST884, ST945, and ST1096 in Saudi Arabia [15]. Another study revealed a detailed molecular analysis of many isolates from Saudi Arabia including the detection of OXA-58 gene in ve isolates and other OXA genes [16]. A number of studies must be undertaken to eliminate extra emergence and distribution of this potent pathogen across the country. Some molecular projects are being processed and designed to study the type of antibioticresistant genes in these strains.

Conclusion
Higher rate of multidrug-resistant A. baumannii bacteraemia has emerged in the southern region of Saudi Arabia as an important health issue. Such a higher prevalence has now been considered as a new threat in the clinical settings, which requires a tremendous e ort to stop its escalation and spread. e occurrence of Acinetobacter strains and the antimicrobial resistance degree continue to rise. e results among the patients, exposed to infections from these organisms, were discovered to be strong enough. Acinetobacter strains also indicated vast challenges for the healthcare facilities, public health, and elderly population.

Consent
Informed consent has been obtained from the participants before starting the procedures.

Conflicts of Interest
e authors declare that they have no con icts of interest.