Detection of NDM and OXA-48 Resistant Genes in Acinetobacter Baumannii Isolated FromIntensive Care Units’ Patients Clinical Samples in Khartoum State

Background: Acinetobacter baumannii is an opportunistic bacterial pathogen with intrinsic and acquired resistance to many antibiotics causing high rates of morbidity and mortality. This study was aimed to detect MDR Acinetobacter baumannii and its resistant genes (bla NDM , bla OXA48 ) from clinical isolates in Khartoum state. Method: A cross sectional hospital-based study was done during the period fromApril to July 2019. A total of 50 clinical isolates were obtained from samples of patients in intensive care units (ICUs) for the purpose of molecular conrming of A. baumannii and detecting NDM and OXA-48 resistance genes by usingconventional PCR. Results: Out of 50 isolates investigated PCR was conrmed 47 (94%) as A. baumannii isolates , while 3(6%) isolates were appeared to be other species. Moreover,the 47 A. baumannii isolates were examined for the presences of resistant genes and the result showed that NDM gene was detected in 2 isolates (4.3%) and OXA-48 gene was detected in only one isolate (2.1%). Conclusion: There is low prevalence of NDM and OXA-48 Resistant Genes among ICUs A. baumannii isolates. However, continuous regional antimicrobial resistance surveillance and improved infection control measures are required in Khartoum hospitals ICUs to prevent further dissemination.


Background
Genus Acinetobacter is a belong to the family Moraxellaceae in the order Pseudomonadales. More than 25 species within the genus Acinetobacter have been described. 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. It possesses little risk to healthy individuals, but generally causes infections in those with weakened immune systems speci cally, the intensive care unit (ICU) [1].
Genus Acinetobacter contains Gram-negative cocco-bacilli which are aerobic, non-fermentative, nonmotile, catalase positive, oxidase negative and with a G + C content of 39-47%. Four species of Acinetobacter including A. calcoaceticus, A. baumannii, A. pittii and A. nosocomialis are similar to each other and it is di cult to distinguish them by phenotypic characteristics. A. baumannii is the commonest species isolated from human clinical specimens, followed by such species as A. luffy, A. pittii, A. nosocomialis, A. haemolyticus and A. johnsonii [2,3].
The clinical impact of Acinetobacter infection in terms of morbidity and mortality has been discussed widely in which the mortality rates range from 19-54%. 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. The clinical signi cance of A. baumannii has grown signi cantly 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 in ux of antibiotics which lead to failure of treatment [1].
Polymyxins are well-established antibiotics that have recently regained signi cant interest as a consequence of the increasing incidence of infections due to multidrug-resistant gram-negative bacteria.
Polymyxin B and Colistin are being seriously reconsidered as last-resort antibiotics in many areas where multidrug resistance is observed in clinical medicine. In parallel, the heavy use of polymyxins in veterinary medicine is currently being reconsidered due to increased reports of polymyxin-resistant bacteria.
In 2009, a novel MBL (metalo-beta-lactam), the New Delhi MBL (NDM), was described. NDM was rst recognized in a K. pneumonia isolate from a Swedish patient who had received medical care in India and was soon recognized as an emerging mechanism of resistance in multiple species of Enterobacteriaceae in the United Kingdom [5]. However, OXA-48-type carbapenem-hydrolyzing class D β-lactamases are increasingly reported in enterobacterial species. To date, there are six OXA-48-like variants have been recognized, with OXA-48 being the most widespread [6].
Acinetobacter baumannii has emerged as a major cause of healthcare-associated infections [1]. It commonly presents resistance to multiple antimicrobial agents, occasionally including carbapenems and polymyxins. Polymyxins are often last-line therapeutic agents used to treat infections caused by multidrug-resistant (MDR) A. baumannii. MDR A. baumannii is a rapidly emerging pathogen, especially in the intensive care setting, causing infections including bacteremia, pneumonia, meningitis, urinary tract infection and wound infection [4] Hence healthcare facilities services are poor in Sudan and there are no recent published studies performed concerning detection of MDR Acinetobacter baumannii resistant genes (NDM, OXA48) this study was performed.

Study design and setting
A cross sectional study was performed during the period from April to July, 2019, in eleven different hospitals in Khartoum, Sudan. 50 Acinetobacter baumannii Isolates obtained from clinical samples were con rmed using polymerase chain reaction (PCR) with a speci c primer for Acinetobacter baumannii (Ac_bum) and for the resistant genes (NDM, OXA48).

Inclusion and Exclusion Criteria
All the clinical isolates obtained from ICU patients' samples and contain Acinetobacter baumanii resistant to commonly used antibacterial were included in this study. However, Acinetobacter baumanii isolated from other hospital departments patients were excluded from this study.

Specimen Collection and Processing
Specimens were ready isolated, identi ed and antibiogram known MDR Acinetobacter spp. Theses isolates were processed through PCR in order to con rm identi cation of A. baumannii and to determine the presence of NDM and OXA-48 genes.
Preservation of the isolates: The bacterial isolates from clinical samples were identi ed and preserved using a sterile loop in 15% glycerol brain heart infusion, charcoal in cryo tubes by placing 2 to 3 colonies and placed in freezer in -20°C until use. Each bacterial isolate holds a speci c number, which is assigned to handle later on. For bacterial recovery from the preservative media; cryo tubes were left to melt in room temperature, a loop full sterile loop is used to streak the suspension in a Muller and Hinton at 37°C for 24 hours.

DNA Extraction:
Whole-cell DNAs were extracted from clinical isolates and standard strains by boiling extract procedure, using a few colonies of each bacterial strain re-suspended in 100 μl of DEPC water. After heating at 100°C for 10 minutes, freezing at -80°C for 10 minutes and boiling for ve additional minutes, the suspensions were centrifuged (5 min, 10,000 ⨉ g) and recovered supernatant was frozen at -20°C until use [7].

Conventional PCR:
Conventional PCR ampli cation for identi cation of Acinetobacter baumannii and for the detection of following genes (NDM, OXA48) was performed by using Maxime PCR premix master mix tube. The primers were designed to amplify internal fragment with product size 791bp (A. baumannii-F and R), 389bp (OXA48-F and R) and 597 bp (NDM-F and R) 380bp table (1) Conventional PCR procedure Reaction mixture ampli ed at the following temperature: initial denaturation at 94° for 2 minutes, 35 cycle of denaturation at 94° for 30 seconds, annealing for 56° for 50 seconds and extension at 72° for 50 seconds. The nal extension at 72° for 50 seconds. Then product has been subjected to gel electrophoresis procedure to detect band size791bp, 597 bp and 380 pb for A. baumannii, OXA48 and NDM genes respectively.

Statistical analysis
Data obtained in this study was analyzed by using SPSS version 20, descriptive analysis were used to describe isolates distribution and frequency and percentage of resistant genes. result has been presented in form of tables and gure.

Results
In this study we include 50 isolates of A. baumannii obtained from patients in different Khartoum state hospitals ICUs. The distribution of isolates according to the samples were 28 (56%) from sputum samples, 6 (12%) from Endo-tracheal tube samples. 5 (10%) for blood and wound swabs samples. While only tow isolates were obtained from urine culture (4%). And one isolates (2%). was originated from CSF, catheter tip and body uid culture respectively [ Table 2].
PCR con rmation of isolates illustrate that frequency of A. baumannii was found to be 47 (94%) while 3 (6%) were negative (other Acinetobacter spp) [ Table 3] [Fig. 1]. NDM gene was detected in 2 (4.3%) out of 47 A. baumannii PCR con rmed clinical isolates, the two isolates were obtained from sputum and wound swab samples [ Table 3] [ Fig. 2]. On the other hand, OXA-48 gene was detected in only one isolate (2.1%) out of the 47 con rmed isolates, and this isolate was obtained from sputum sample [ Table 3] [Fig. 3].

Discussion
Recently, A. baumannii has become a major hospital pathogen especially in ICUs. Various factors, including poor immune system, consumption of antibiotics, clonal spread of resistant microorganisms, poor infection control and drug resistance mechanisms, result in the dissemination of highly resistant pathogens to commonly used antibiotics [1].
In the present study 50 clinical isolates were collected for the purpose of identifying A. baumannii and detecting NDM and OXA-48 resistance genes by using PCR. In the present study, sputum showed the highest frequency among other type of samples with 28 (56%), which is similar to the result found by nding in 2013 was disagreed with our nding, the found in a total of 150 A. baumannii isolates, sputum showed the greatest frequency among other type of samples 77 (51.3%) [9].
In term of molecular con rmation, the frequency of A. baumannii was found in the present study was 47 (94%) which is similar to the frequency rate found out by Falah etal., in 2019 whom con rmed 80 (97.56%) of A. baumannii isolates in a total of 82 [2]. While Marathe et al., in 2019 found results which were strongly disagreed with the frequency found in the present study they examined a total of 112 sample and found only 33(30%) con rmed as A. baumannii [10].
In this study, NDM gene was detected in only 2 isolates (4.3%) out of 47, while Marathe et al., in 2019 detected 29 (87.8%) NDM in a total of 33 Albuminoid, which is dramatically high result compared to the frequency rate of NDM found in the present study. Also, Karaaslan et al., in 2016 found high frequency rate of NDM among A. baumannii 22 (31%) in a total of 72 samples [11]. Bakour et al., in 2015 showed frequency rate of NDM 10 (22.7 %) in a total of 44 A. baumannii which is also considered high according to the frequency of NDM found is the present study [12]. Khorsi etal., in 2015 found 10 (10.6 %) NDM A. baumannii in a total of 94 sample which is moderate results compared to the above results [13]. While Howard etal., in 2012 found 2 (1.85) NDM-1-producing Acinetobacter baumannii isolates out of 108 sample which is lower frequency rate compared to the above results and approximately close to the frequency rate found in our study [14]. This discrepancies between our nding and the previous studies may be due to variation in sample size and study populations.
In this study OXA-48 gene was detected in only one sample (2.1%) out of 47 sample. Study of Robustillo-Rodela etal., in 2017 found that 13 patients were colonized or infected by OXA-48 out of 31 which disagreed with our nding. The cumulative incidence of OXA-48 was 3.48% which is considered high frequency rate compared to the ndings of the present study [15]. While Bakour et al., in 2015 found no OXA-48 gene among 44 isolates which is closer to the results found in this study [12]. This discrepancies between our nding and the previous studies may be due to variation in sample size and study populations.