2.1 Study site and period
This cross sectional, hospital-based study was conducted at Annapurna Neurological Institute and Allied Science (ANIAS) Hospital and Annapurna Research Centre (ARC), Maitighar, Kathmandu, Nepal over a six-month period from February to August 2018.
2.2 Sample description
A total of 380 different clinical specimens including urine, blood, sputum, catheter tip, cerebrospinal fluid (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 blaOXA−23 gene was the dependent variable.
2.3 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 fluid, 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 identification 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 identification tests were performed. All the inoculation and media preparation works were strictly performed under the laminar flow cabinet to prevent contamination.
2.4 Phenotypic identification 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 identified 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 identification of Acinetobacter spp. was done by standard laboratory procedure. After overnight incubation, typical non-fermenting colonies of Acinetobacter spp. were identified. These colonies were then subjected to further processing via gram staining and other recommended biochemical tests [16]. A. baumannii was identified 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, H2S, and gas negative in TSI test [17].
2.5 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. Identified 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 Modified 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.
2.6 Preservation of the A. baumannii isolates
After the AST, the confirmed 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 Modified Hodge Test (MHT) and molecular tests.
2.7 Modified 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].
2.8 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 blaOXA−23 gene was performed using specific primers (F: 5ʹ-GATCGGATTGGAGAACCAGA-3ʹ, B: 5ʹ-ATTTCTGACCGCATTTCCAT-3ʹ) ((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 final extension at 72 °C for 10 minutes. PCR products were examined using 1% agarose gel electrophoresis containing 0.5 µg/ml ethidium bromide [11].
2.9 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.
2.10 Statistical analysis
All the results were entered in the worksheet of Statistical Package of Social Sciences (SPSS 16.0). Chi-square test was used to determine the association of independent variables. A value of α ≤ 0.05 will be assumed wherever applicable and 95% confidence intervals along with the exact p-values will be presented.