Molecular Detection of blaoxa-23 Gene from Carbapenem Resistant Acinetobacter Baumannii Isolated from a Tertiary Care Hospital of Nepal

Background: Infections associated with Acinetobacter baumannii are increasing in many parts of the world especially the healthcare associated infections (HAIs). Antibiotics resistance is a great concern of public health which is either an inherent or adaptation property of microorganisms to resist the action of antibiotics. Carbapenem resistance, mainly among gram-negative bacteria is an ongoing problem that causes serious infections and dramatically limits the treatment alternatives. The prospective cross-sectional study was designed to detect the bla OXA-23 gene from carbapenem-resistant A. baumannii isolates in a tertiary care hospital of Nepal. Methods: A total of 380 clinical specimens (tracheal aspirate, urine, sputum, blood and wound samples, Foleys tips, and catheter tips) were collected from the study population and were examined by microbiological procedures including Gram’s staining, culture, and various biochemical tests. Antibiotic susceptibility testing (AST) was done as per the protocol of Kirby-Bauer disk diffusion technique and the CLSI guidelines while screening of carbapenemase production was checked through Modied Hodge Test (MHT) using Meropenem (10µg) disc. All the phenotypically positive results for carbapenemase production were further analysed by PCR and agarose gel electrophoresis for molecular detection of the bla OXA-23 gene. Results: Among 380 specimens analysed, 55.3% (210/310) of samples were positive for bacterial growth where 15.7% (33/210) of bacterial isolates were A. baumannii and 69.7% (23/33) were carbapenem-resistant. High prevalence (21.2%) of A. baumannii was among the patients of age group 51-60 followed by 41-50 years (18.2%) years but the result was statistically insignicant (P>0.05) and more isolates were from ICU (60.6%) followed by post-operative patients (18.2%) The bla OXA-23 carbapenemase gene was found in 82.6% (19/23) of meropenem resistant isolates while 97% isolates of A. baumannii were susceptible to colistin. Conclusion: The high rate of antibiotic resistance is funnelling the therapeutic options for the treatment of infections associated with A. baumannii which clearly shows a need for rational use of antibiotics. Systematic network surveillance should be established for monitoring and controlling the spread of the antibiotic-resistant gene of pathogenic bacteria especially in a resource-limited clinical setting like Nepal.


Introduction
The number of nosocomial infections caused by Acinetobacter baumannii is gradually increasing in recent years especially, more concern is in critically ill patients who are highly liable to infections and the risk factors for the infection are not well established [1]. The ubiquitous distribution, survival ability and high rate of resistance to the commonly used antibiotics are responsible for the emergence of A. baumannii as a signi cant nosocomial and opportunistic pathogen and have created a greater threat and challenge to treat the infections associated to it [2]. A. baumannii is considered as one of the frequently isolated opportunistic clinical pathogens and due to the tendency to acquire mechanism of antimicrobial resistance it can cause community as well as healthcare-associated infections (HAIs) [3]. Since the early 1970s, multidrug-resistant (MDR) strains of Acinetobacter have been reported and A. baumannii is also associated with some of the disasters and con ict as a cause of osteomyelitis and wound infections like in 2002 Bali army operations, and earthquake of Turkey in 1999 [4]. The infections associated with A. baumannii range from pneumonia to meningitis but, more infections are reported from organs containing a high level of body uids such as respiratory tract, peritoneal cavity, and urinary tract. In the majority of cases, it is believed that infections are developed after getting in contact with the hospital equipment contaminated by A. baumannii [5] which leads to severe illness with the high rate of patients death of about 30% [6]. Hospital-acquired infections like pneumonia, bloodstream and burn infections, meningitis, soft tissue infections, and osteomyelitis has made A. baumannii as a major potent and troublesome pathogen of health care setting and world health organization (WHO) has listed carbapenem-resistant A. baumannii as a priority pathogen of the 'Critical Group A' which needs new antibiotics to address the increasing antibiotic resistance (5,7). Due to the unique ability to acquire or adapt antimicrobial resistance features A. baumannii is being a threatening pathogen to our antibiotic-based treatment strategy. Multiple number of genes are reported from A. baumannii which have role in attachment to abiotic surfaces and development of bio lm [8]. Multidrug resistant (MDR) strains have been isolated worldwide and it has been demonstrated that these strains can spread from area with high rates of antimicrobial resistance to other areas with historically low rates [9].
Basically, antibiotics of carbapenem group are considered as reserve drug for the treatment of multiresistant A. baumannii caused infections but carbapenem-resistant strains of A. baumannii have been reported from multiple places [10]. A. baumannii can be resistant to carbapenem by various means but, mainly it is determined by hydrolysis of antibiotics by bacterial enzymes especially carbapenemhydrolysing β-lactamases group of enzymes like oxacillinases which has six-subtypes which correspond to class D acquired bla OXA−23 -like (OXA-23, 27, and 49), chromosomal blaOXA-51-like, blaOXA-24/40-like (blaOXA-24-26, 40 and 72), blaOXA-58-like, blaOXA-235-like (OXA-235 to 237) and blaOXA-143-like enzymes and several antibiotic resistance cases due to production of acquired blaOXA have been identi ed in multiple places while A. baumannii carrying the bla OXA−23 -like gene is worldwide in distribution (11,12,13). Some of the insertion sequence (IS) elements like ISAba1, ISAba2, ISAba3, ISAba4 and IS18 have a positive role for the development of carbapenemases genes in A. baumannii and ISAba1 can spread the carbapenemases genes among the species of Acinetobacter [14]. Wider distribution of antibiotic resistance blaOXA−23 -like gene has put the modern therapeutic options at a greater risk which has increased the hospital stay of patients with severe form of illness leading to economic and societal burden. Despite frequent antibiotic resistance cases being reported in Nepal there is very limited data on prevalence of bla OXA−23 -carrying A. baumannii so, this study was designed with an aim to determine the antibiotic susceptibility patterns and report the data on bla oxa−23 gene among the carbapenem resistant A. baumannii isolates from a tertiary care hospital of central Nepal.

Sample description
A total of 380 different clinical specimens including urine, blood, sputum, catheter tip, cerebrospinal uid (CSF), tracheal aspirates, and central venous catheter (CVP) tips were collected from different wards of hospital. Specimens were from patients of general ward, post-operative ward and intensive care unit (ICU) of the hospital. A pre-formed questionnaire was used to record the patients' clinical and demographic data. Ethical approval for this study was given by the ethical review committee of National Health Research Council (NHRC), Ramshah Path, Kathmandu, Nepal (ref. no. 388/2018). Well-informed written consent was taken from all the enrolled patients in local language before each sample collection this article does not contain any individual patient data. For this study, age, sex, and antibiotic resistance were considered as independent variables while bla OXA−23 gene was the dependent variable.

Collection and culture of samples
The specimens collected for this study were urine, blood, sputum, catheter tip, CSF, tracheal aspirates, and CVP tips. Each specimen was separately collected in a sterile container. All the specimens were processed and analysed in the microbiology laboratory of the hospital and PCR along with gel electrophoresis was performed in the ANIAS Research laboratory, Kathmandu, Nepal. Specimens like cerebrospinal uid, urine, tracheal aspirates, and sputum were inoculated on the MacConkey agar (MA) and Blood agar (BA) plates then incubated at 37℃ for 24 hrs. After 24 hours of incubation at 37℃ plates were observed for the growth and further analysis. For all the blood specimens, blood in BHI (Brain Heart Infusion) broth was incubated aerobically for at least 7 days at 37℃. Each day one loopful of the inoculated broth was inoculated in MA and BA plates which were incubated at 37℃ overnight, growth on plates was observed after the incubation period and processed accordingly for the identi cation of bacterial strains. Other specimens like catheter tips, CVP tips, and Foley's tips were held with sperate sterile forceps and rolled over the surface of MA and BA plates then plates were incubated at 37℃ for overnight. After incubation, the growth of isolates was observed, and further identi cation tests were performed. All the inoculation and media preparation works were strictly performed under the laminar ow cabinet to prevent contamination.

Phenotypic identi cation of the bacterial isolates
After 24 hours of incubation, visual growth on the inoculated plates was observed and colony morphology was noted. The isolated colonies were then identi ed as per the morphology, gram staining and various biochemical tests which were catalase test, oxidase test, Voges Proskauer test, Methyl Red test (MR-VP), citrate test, urease test, oxidative-fermentative test, TSI (Triple Sugar Iron agar) test [15]. The identi cation of Acinetobacter spp. was done by standard laboratory procedure. After overnight incubation, typical non-fermenting colonies of Acinetobacter spp. were identi ed. These colonies were then subjected to further processing via gram staining and other recommended biochemical tests [16]. A. baumannii was identi ed after performed series of biochemical tests such as positive catalase and citrate test, negative oxidase and urease test, non-motile, indole negative, oxidative in Hugh and Leifson's medium, negative gelatin hydrolysis test, acid production from glucose, lactose, xylose, galactose, mannose but not from sucrose and mannitol and ability to grow at both 37℃ and 44℃, alkaline slant/alkaline butt i.e. glucose, lactose and sucrose non-fermenter, H 2 S, and gas negative in TSI test [17].

Antibiotic susceptibility tests (AST) of A. baumannii
The AST test was performed as per the protocol of Kirby-Bauer disc diffusion technique to check the sensitivity and resistivity of A. baumannii isolates against the antibiotic discs. Identi ed strains of A. baumannii were inoculated on sperate Mueller-Hinton agar (MHA) plates and their susceptibilities to carbapenem were tested by disc diffusion method according to the guidelines of Clinical and Laboratory Standard Institutes (CLSI -M100-S25, 2015) [18]. Inoculum were prepared by suspending the single isolated colony on the nutrient broth and comparing the turbidity with 0.5 McFarland standards after proper incubation time. Carpet culture of bacterial suspension were performed on the MHA plate. After antibiotic discs were kept on the agar plate using sterile forceps then time was provided for the proper diffusion. Finally, plate was incubated for 18 hours at 37 °C. The diameter of zone of inhibition (ZoI) was measured for around all the discs then results were interpreted as recommended by CLSI guidelines and isolates were reported as 'resistant', 'intermediate' and 'sensitive' (Appendix-A). In this study, 12 antibiotic discs of different classes were used for antibiotic susceptibility test. We have used carbapenem class of antibiotics (Ertapenem-10 µg, Meropenem-10 µg, Imipenem-10 µg) to check the carbapenemase resistance among the A. baumannii isolates. As per the CLSI protocol, A. baumannii isolates which has shown susceptible or intermediate zones on AST for imipenem disc (16-21 mm) were further tested by Modi ed Hodge Test for phenotypic detection of carbapenemase production [19]. Antibiotics and quantity were selected based on prescription frequency by physician and availability at the time of study.
Minimum inhibitory concentration (MIC) of the tested antibiotics were not determined due to unavailability of antibiotics powder and limited research fund.

Preservation of the A. baumannii isolates
After the AST, the con rmed carbapenem-resistant A. baumannii isolates in pure culture were preserved in 20% glycerol containing Tryptic Soya broth and kept at -70 °C until subsequent tests like Modi ed Hodge Test (MHT) and molecular tests.

Modi ed Hodge Test
It is a phenotypic screening test which is used to identify the carbapenemase producers. For the MHT, 0.5 McFarland dilution of the Escherichia coli ATCC 25922 in 5 ml of broth was prepared. Then it was diluted 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of saline. A lawn of the diluent was streaked on MHA and left to dry for 3-5 minutes. Then 10 µg meropenem/ertapenem antibiotic disc was placed at the centre of the plate. Then after, A. baumannii isolates were streaked straight from one edge of the disc to the edge of the plate at 3 different places keeping the equal gap between them and plates were incubated for 24 hours at 35 °C in presence of ambient air. After the incubation period, clover leaf-type depression at the intersection of E. coli 25922 and A. baumannii was MHT positive while there was no growth of E. coli 25922 along the test isolates growth streak on the antibiotic disc diffusion area [20].

Molecular examination
2.8.1 Crude plasmid DNA extraction of A. baumannii and PCR reaction Carbapenem resistant A. baumannii was preserved on Tryptic soya broth for plasmid DNA extraction process. DNA was extracted by alkaline hydrolysis method in which A. baumannii strain was cultured in LB (Luria Bertani) broth at 37 °C for overnight as describe previously [21]. The amount of extracted DNA was examined by spectroscopy at 260 nm. PCR reaction to identify bla OXA−23 gene was performed using speci c primers (F: 5 -GATCGGATTGGAGAACCAGA-3 , B: 5 -ATTTCTGACCGCATTTCCAT-3 ) ((Primers used reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613023/) [22,23]. The cycling conditions were performed with a preliminary denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, and annealing at 52 °C for 40 s, with a nal extension at 72 °C for 10 minutes. PCR products were examined using 1% agarose gel electrophoresis containing 0.5 µg/ml ethidium bromide [11].

Quality Control
Mueller Hinton agar and the antibiotic discs were checked for their lot number, manufacture and expiry date, and proper storage. For the standardization of Kirby-Bauer test and for performance testing of antibiotics and MHA, control strains of Escherichia coli (ATCC 25922) were tested primarily. Quality of sensitivity test was monitored by maintaining the thickness of Mueller-Hinton agar at 4 mm and the pH at 7.2-7.4.

Statistical analysis
All the results were entered in the worksheet of Statistical Package of Social Sciences (SPSS 16.0). Chisquare test was used to determine the association of independent variables. A value of α ≤ 0.05 will be assumed wherever applicable and 95% con dence intervals along with the exact p-values will be presented.

Bacterial growth
A total of 380 clinical specimens from in-patients (ICU ward, general ward, post-operative ward) were received and processed in the microbiological laboratory of ANIAS and Annapurna research centre during the study period. Among 380 analysed specimens, 55.3% (210/380) have shown aerobic bacterial growth while 44.7% (170/380) specimens were negative for the bacterial growth. Out of 210 culture positive, 15.7% (33/210) isolates were con rmed as A. baumannii by microscopic observation of gram stain, series of biochemical tests, and growth temperature (Fig. 1).

Comparative evaluation of carbapenemase production by phenotypic tests
Two phonotypic tests AST and MHT were used to identify the carbapenemase producing A. baumannii where 87.9% (29/33) of isolates were screened positive for carbapenemase production by Kirby-Bauer disc diffusion where 69.8% (23/33) of isolates were con rmed as carbapenemase producer by Modi ed Hodge Test. Out of 29 carbapenem resistant A. baumannii by AST, 79.3% (23/29) of the isolates was found to be MHT positive while 20.7% (6/29) was found to be negative and the result was statistically signi cant (p = 0.001) ( Table 4).   Table 6).

Discussion
Emergence and spread of drug resistant A. baumannii have been recognized as a potent pathogen which has unique ability to survive in a hospital environment and remain for a long period of time and possess a great public health concern which is highly associated with HAIs [24]. respectively [29,30].
In our study, the highest number of A. baumannii isolates was found in sputum (48.5%) followed by wound swab (24.3%), tracheal aspirates (12.1%), urine (9.1%), blood (3%) and catheters tip (3%) respectively where the result was not statistically signi cant (P = 0.310). In agreement with this nding, Jaggi et al. (2012) have found maximum isolates from respiratory secretions (57.4%) followed by blood (23.8%) in India [31] and similarly highest isolates were reported in respiratory tract specimens in study conducted by Yadav et al. (2020) and Shrestha et al. (2016) from Nepal [32,33]. This study highlights the higher prevalence of A. baumannii among ICU patients (60.6%) followed by post-operative (21.2%) and patients of general ward (18.2%) respectively. This result supports the role of A. baumannii as a major cause of ventilator-associated pneumonia (VAP) especially among critically ill patients and recent studies shows the mortality rate of hospital patients from MDR-AB ranged from 52-66% and the high rate of MDR-AB transmission among ICU patients is very common mainly in resource limited setting [34] [32]. Low rate of A. baumannii susceptibility towards the third and fourth classes of cephalosporin antibiotics is associated with the ESBL and AmpC β-lactamase positive strains. In essence, OXA-type carbapenemases are common in A. baumannii and the acquired bla OXA−23 gene is the main genetic element in Asian countries. The worldwide emergence of CRAB is a matter of concern to clinician which is mostly associated with grade D β-lactamases and MBLs. Plasmid carrying bla OXA−23 gene can be transported within the strains of A. baumannii via conjunction so, MDR and CRAB are speedily growing globally [35]. Other OXA-type genes like bla OXA−24 and bla OXA−58 are more common in isolates of A. baumannii from European region [36]. Other novel class D β-lactamases genes like bla OXA−143 and bla OXA−235 have been reported from different places of the USA, Mexico and Brazil [37,38]. The CRAB is listed as a global priority pathogen in critical group by WHO which needs urgent efforts for the development of new antibiotics for the treatment of CRAB [7]. We have compared the two phenotypic tests; AST by Kirby-Bauer and MHT for the screening of carbapenemase producer A. baumannii where 87.9% (29/33) isolates were positive for carbapenemase production by Kirby-Bauer disk diffusion technique while 69.8% (23/33) isolates were carbapenemase producer by MHT and the result was statistically signi cant (P = 0.001). All the MHT positive isolates were assessed for the detection of bla OXA−23 gene using conventional PCR and all MHT positive isolates for carbapenemase production were positive for the target gene and the result was statistically signi cant (P < 0.05). Carbapenemase production was evaluated by different phenotypic techniques including MHT while the genotypic nding of carbapenemases among CRAB isolates using PCR is the most convincing [19]. Identi cation of carbapenemases among MDR strains of A. baumannii is crucial to plan the therapeutic regimen for clinician because CRAB limits the treatment options and can cause more deaths of infected patients. The production of carbapenem-hydrolysing class D β-lactamase (CHDLs) and MBL are the most common mechanisms which play positive role in carbapenem resistance [39]. The sensitivity of the MHT in our study (82.6%, 19/23) is in agreement with other studies reported previously where MHT has detected carbapenemase producer in 83.3%, and 73% among the screened isolates [40,41]. As per CLSI 2018 guidelines, MHT is not recommend for carbapenemase detection as a phenotypic test which could be due the poor speci city of the MHT when con rming some ESBL production happening with porin loss [42]. Written consent was taken from all the patients in local language before sample collection. This manuscript does not contain any individual human or animal data.

Consent for publication:
Not applicable Availability of Data and Materials: The datasets used and analysed during this study are available in excel sheets which can be obtained from the corresponding author on reasonable request.
Competing Interests: The authors declare they do not have any competing interests. Table   Table 6 not available with this version. Figure 1 Prevalence of A. baumannii among culture positive bacteria Gel Electrophoresis of PCR amplicons of blaOXA-23 gene (Sample 1 to 5)