Study design and study sample
This cross-sectional study was conducted from March to August, 2019 at Kathmandu Model Hospital, Kathmandu, Nepal. A total of 3216 clinical samples consisting of urine (n=1776), blood (n=875), pus (n=156), sputum (n=187), body fluids (n=88), wound swab (n=51), tissue (n=18), catheter and other tips (n=12), and other samples including stool, urethral and vaginal swabs, bone, and bone marrow aspirate (n=53) was collected and processed during the study period. Patients of all age-groups and genders who were admitted in or visiting the study hospital (for treatment) were included in this study. All the samples with completely filled demographic information having no visible signs of contamination were included in the study. However, others were rejected and requested for repetition, if possible.
Sample Collection and transport
All the samples were aseptically collected following the standard microbiological procedure. Individual collection procedures varied in accordance to the type of samples. Generally, samples were collected in a dry, wide-mouthed, and leak-proof container and were sent to microbiology department without delay. In case of unwarranted delay, clinical specimens were refrigerated at 4°C to 6°C.
Culture, isolation and identification of bacteria
Each sample was processed by following the standard microbiological guidelines [29, 30].
Urine sample: Urine samples were inoculated into Cysteine Lactose Electrolyte Deficient (CLED) agar using sterile and standard calibrated loop. The plates were incubated at 37°C overnight.
Blood and (endotracheal and catheter) tips: Blood specimen was inoculated aseptically onto BHI broth at the ratio of 1: 10 and was incubated at 37°C for 7 days and routinely inspected twice a day for at least first three days for microbial growth. Then broth from the culture bottle showing visible signs of microbial growth was sub-cultured in Blood agar (BA), MacConkey agar (MA) and Salmonella-Shigella agar (SS) and plates were incubated at 37°C for 24 hrs [31].
Sputum and throat swab: Sputum samples were inoculated into BA, CA and MA plates. For sputum, in CA plate a 5 μg optochin disc and a 10U bacitracin disc were added to screen out S. pneumoniae and H. influenzae respectively whereas for throat swab, 0.05U bacitracin disc was added to the plate to screen Streptococcus pyogenes. Chocolate agar and BA were incubated at 37°C overnight in 5-10% CO2 environment whereas the MA plate was incubated at 37°C in an aerobic condition [31].
Pus, pus swab, and wound swab: These samples were inoculated into BA and MA plates, and inoculated at 37°C for 24hrs. In case of swab, an initial inoculum was made by rubbing the swab over the media plate in order to transfer maximum number of organisms. Then the streaking was performed [31].
Body fluids: Body fluid samples were centrifuged before culture. The sediment after centrifugation was inoculated into BA, MA and CA plates. The BA and CA plates were incubated in a 5-10% CO2 enriched atmosphere and MA plates were incubated aerobically at 37°C overnight [31].
Identification of the isolates
Following incubation, culture plates were observed for possible microbial growth. Isolates were presumably identified on the basis of Gram’s staining and colony characteristics. Further confirmation of the isolates were based on biochemical tests such as IMViC (Indole production, Methyl red test, Voges-Proskauer test and Citrate utilization), H2S production, catalase test, coagulase test, and oxidase test [30, 32].
Antibiotic susceptibility test (AST)
Isolates were further subjected to in-vitro antibiotic susceptibility assay by using modified Kirby-Bauer disk diffusion method as recommended by Clinical Laboratory Standard Institute [33]. Nitrofurantoin (300µg), cefotaxime (30µg), cotrimoxazole (25µg), cefixime (5µg), amoxycillin (10µg), ofloxacin (5µg), levofloxacin (5µg), gentamicin (10µg), moxifloxacin (5µg), ceftazidime (30µg), amoxycillin/clavulanate (20/10µg), amikacin (30µg), ciprofloxacin (5µg), chloramphenicol (30µg), azithromycin (15µg), cefoperazone/sulbactam (75/30µg), meropenem (10µg), imipenem (10µg), ertapenem (10µg), piperacillin/tazobactam (100/10µg), doxycycline (30µg), cefepime (5µg), ampicillin/sulbactam (10/10µg), polymyxin-B (100µg), colistin (10µg), and tigecycline (15µg) discs were tested for susceptibility assay. Each antibiotic disc consisted of the aforementioned amount of antibiotics as per the type of antibiotic disc used. In this method, broth culture of test organism (comparable to McFarland tube no.0.5; inoculums density 1.5×108 organisms/ml) was uniformly carpeted on the surface of Mueller Hinton agar (MHA). Then, antibiotics discs were placed onto the lawn culture of the test organism by sterile forceps. The inoculated and seeded MHA plates were then incubated at 37° C for 24 hours. After incubation, results were interpreted as sensitive, intermediate and resistant [33]. Isolates showing resistance to at least one agent of three or more classes of antimicrobial agents were termed as multidrug-resistant (MDR) [34].
Screening of the ESBL production
ESBL-producers were screened by using Ceftazidime (30µg) and Cefotaxime (30µg) in the AST. Isolates showing reduced susceptibility to one or both of these drugs with diameter of the zone of inhibition (ZOI) for ceftazidime ≤22 mm and cefotaxime ≤27 mm were considered as potential ESBL-producers [33]. Suspected strains were further processed for confirmatory assay.
Confirmation of the ESBL-producers by phenotypic method
Combination disc test (CDT) as prescribed by the CLSI was the choice of protocol for confirmation of the ESBL-producing strains. In this method, cefotaxime (30µg) and ceftazidime (30µg) discs alone and in combination with clavulanic acid (10µg) ( ceftazidime plus clavulanic acid, 30/10 µg and cefotaxime plus clavulanic acid, 30/10 µg) were used. The ZOI of cephalosporin discs alone was compared with their respective cephalosporin/clavulanic acid (combined) discs. An increase in ZOI by ≥5mm in the presence of clavulanic acid was considered as confirmed ESBL production test [33].
Screening for carbapenemase and/or KPC producers
In AST, isolates showing resistance to carbapenem drugs (imipenem 10µg, meropenem 10µg, and ertapenem 10µg) were suspected as potential carbapenemase-producers [27].
Phenotypic confirmatory test for carbapenemase and/or KPC producers
Inhibitor-based method was followed for the confirmation of carbapenemase and KPC production. In this method, combined disc test of carbapenem with and without PBA was employed. An increase in the diameter of ZOI by ≥ 5mm in combined disc (carbapenem disc supplemented with PBA) than single disc (only carbapenem disc) was considered as confirmed test for carbapenem or KPC production [35, 36].
Phenotypic confirmatory test for MBL production
Similar to carbapenemase production test, confirmation of MBL production was made by inhibition method in which Ethylene Diamine Tetra Acetic Acid (EDTA) was used as an inhibitor. Two imipenem (10µg) discs were placed on MHA and 10µl of 0.5 M EDTA solution was added to one of the discs to obtain the desired concentration. After overnight incubation, an increase in the diameter of ZOI by 7 mm in combined disc (imipenem disc supplemented with EDTA) than single one (only imipenem disc) was considered as confirmed test for MBL production [37].
Determination of MIC of colistin
Agar dilution was used to determine the minimum inhibitory concentration of colistin. In this method, different concentrations of colistin ranging from 2 µg/ml to 32 µg/ml were prepared in the agar medium. Bacterial inoculum was then applied readily onto the agar surface and the plates were incubated at 37°C up to 18 hours. The MIC end point was determined as the lowest concentration of antibiotics that completely inhibits the visible growth. Isolates having a MIC of ≤4 μg/mL is considered colistin susceptible while MIC of >4 μg/mL is considered colistin resistant [22, 38].
Plasmid and genomic DNA extraction
Plasmid DNA of E. coli and K. pneumoniae was extracted by using phenol-chloroform method [39]. Firstly, isolated colonies of bacteria were inoculated in Luria–Bertani (LB) broth and incubated overnight at 37oC in the presence of air. After required incubation, alkaline-lysis method was adopted to extract the DNA. Extracted plasmid DNA was then suspended in TE buffer and preserved at −20°C until further processing [39].
PCR amplification of mcr-1 gene
Amplification of mcr-1 gene was carried out by conventional PCR using primers: 5′-CGGTCAGTCCGTTTGTTC-3′ as forward primer and 5′-CTTGGTCGGTCTGTAGGG-3′ as reverse primer [2]. A PCR mixture having the final volume of 25µl (3µl of template DNA, 0.5µl each forward and reverse primes and 21µl of PCR master mix) was used for the reaction mixture.
The thermal condition for amplification was initial heat activation of 95ºC for 15 minutes followed by 35 cycles of denaturation at 94ºC for 30 seconds; annealing at 57ºC for 90 seconds; extension at 72ºC for 90 seconds; and final extension at 72ºC for 10 minutes. The amplified products were subjected to gel electrophoresis (2.0% agarose gel stained with µl ethidium bromide) at 100V for 60 minutes and visualized under UV transilluminator [22].
Quality control during antimicrobial susceptibility and MIC assays
Each batch of media, reagents and antibiotic discs were checked for their lot number, expiry date, and proper storage. Similarly, purity plates were used to ensure the pure culture of test organisms. Control strain of E. coli ATCC 25922 was used during AST.
Data analysis
All the data were entered in the worksheet of statistical package for social science (SPSS) software (Version 25) and presented in the form of tables and figures. Chi-square (χ2) test was applied to predict the relationship between the variables. A p value of <0.05 was considered as statistically significant.