Extended spectrum betalactamase and Carbapenemase producing gram negative bacteria from healthcare workers gowns at Debre Berhan Comprehensive Specialized Hospital, Ethiopia

Study area, study design, and period
A hospital-based cross-sectional study was conducted from August to October 2022 at Debre Berhan Comprehensive Specialized Hospital (DCSH) in Debre Berhan town, North Shewa Zone, Amhara regional state, Northeast Ethiopia. Debre Berhan town is 130 km far from the country’s capital, Addis Ababa. The hospital served the local community for many decades before being upgraded to a Referral Hospital in 2010. Since 2012, it has been upgraded to a Comprehensive Specialized Hospital. It is the only hospital in the North Shewa Zone of the Amhara region that serves as a referral centre for seven governmental district hospitals and two private hospitals. Furthermore, it has more than 200 beds; it serves over two million people in the Amhara, Afar, and two woredas of Oromia regions. Pediatric surgery, gynecology, psychiatry, ophthalmology, HIV care, and outpatient clinics are among its specialties. The hospital has a total of 645 workers of these 408 are healthcare professionals11.
Source and study population
All healthcare workers who were working at DBCSH were the study participants and all healthcare workers who were involved in clinical services during the study period were the study participants.
Sample size determination and sampling technique
Sample size determination
The sample size was determined using a single population proportion formula by considering the prevalence (P) of a previous study in Addis Ababa (9.4%)12 and as follows:
n = z2p (1-p).
d2.
Where: n = the minimum required sample size;
z = Standard normal distribution value at 95% CI, which is 1.96;
P = the prevalence.
d = the margin of error taken as 4%.
n = z2p (1-p) = 1.962(0.094) (0.906) = 205.
d2 (0.04)2.
The initial sample size plus adding a 10% non-respondent rate, the total sample size was 226.
Sampling technique
A simple random sampling technique was used to select study participants after proportional allocation was applied to each stratum of the occupational group. Study participants were selected using a simple random sampling technique (Fig. 1).
Ni = nNi/N, Where; N = the total population size.
Ni = population size of each occupation.
Ni = sample size drawn from each profession, n = sample size required.

Schematic presentation of sampling techniques based on professions. Others professions include Health officer, midwife, physiotherapy, radiologic technicians, anesthetists, ophthalmologists.
Data collection and laboratory methods
Data collection
Scio-demographic, and gown-related data (hand hygiene practice, gown type, frequency of gown changing, service year, area of working unit/department/ward, laundering practices, frequency of laundering, and wearing gown outside clinical areas) were collected by pre-tested self-administered questionnaire.
Sample collection and transportation
Swab samples were collected from the gown of each HCW using a sterile cotton swab moistened with sterile normal saline. Gown swab samples were also collected in areas that are most exposed to contamination such as around pockets, buttons, hands, and other surface areas. Swab specimens were transported immediately to the microbiology laboratory of DBCSH for bacteriological analysis.
Bacterial culture, isolation and identification
Gram-negative bacteria were isolated on MacConkey agar (Oxoid Ltd. Bashingstore, Hampaire, UK) using calibrated loops and incubated aerobically at 37 0C for 24 h. Cultures that did not grow any colonies after 24 h were further incubated for an additional 24 h. Identification of all the isolates was done using gram staining and biochemical tests, namely indole, citrate, H2S production, lysine decarboxylase, lactose fermentation, urea hydrolysis, gas production, malonate, catalase, and mannitol fermentation. The oxidase test was used to differentiate Pseudomonas and Acinetobacterspp13,14.
Antimicrobial susceptibility test
The Kirby-Bauer disk diffusion method was used to test the antimicrobial susceptibility of antibiotics as per the Clinical Laboratory Standard Institute (CLSI) guidelines15. Bacterial inoculum was prepared by suspending the freshly grown bacteria in 3–5 ml normal saline and turbidity was adjusted to 0.5 McFarland standards. A sterile cotton swab was dipped and rotated several times and was pressed against the wall of the test tube. It was then swabbed over the entire surface of the Mueller Hinton agar (HI Media, India) and antimicrobial disks were applied to the plate. The susceptibility of the isolated gram-negative bacteria was tested against imipenem (IMP:10 µg), meropenem (MEP:10 µg), ampicillin (AMP:10 µg), trimethoprim/sulphamethoxazole (SXT:1.25/1.23 µg), ciprofloxacin (CIP:5 µg), cefotaxime (CTX:30 µg), ceftazidime ( CAZ: 30 µg), amikacin (AMK: 30 µg), ceftriaxone (CRO: 30 µg), cefuroxime (CXM: 30 µg), chloramphenicol (C: 30 µg), tobramycin (TOB: 30 µg ), (amoxicillin- clavulanic acid (AMC:30 µg), Tetracycline (TET: 30 µg) and gentamicin (GEN:10 µg). The results were interpreted as susceptible, intermediate, or resistant as per the standard.
Screening of ESBLs and carbapenemase
Screening of ESBL and carbapenemases was done by chrome-ESBL and carbs-chrome agar (Liofilchem®, Italy). The test organisms were inoculated on a chromogenic agar plate using the spread plate technique of direct streaking and incubated at 37oC for 18–24 h. Colonies of ESBL and carbapenemase producers produce species-specific colors.
Phenotypic confirmation of ESBLs with combination disc test
A disc containing 30 µg of ceftazidime and 30 µg of cefotaxime, alone, as well as their combination with clavulanic acid (30 ug/10 ug) acid, were placed at a distance of 25 mm, center to centre, on an MHA plate that has been seeded with a bacterial suspension of 0.5 McFarland turbidity standard, and then incubated overnight (18–24 h) at 37 °C. Gram-negative bacterial isolates with an increase in inhibition zone diameter of greater than or equal to 5 mm for a combination disc versus ceftazidime or cefotaxime disc alone were identified as ESBL producers15.
Phenotypic confirmation of carbapenemase production
Gram-negative bacterial isolates that were not susceptible to imipenem or meropenem were tested for the presence of carbapenemase using the CLSI-recommended modified carbapenem inactivation method (mCIM).No zone inhibition of the meropenem susceptible strain (E. coli ATCC 25922) around the meropenem disk, zone of diameter 6–18 mm or the presence of pinpoint colonies within a 16–18 mm zone of susceptible strain (E. coli ATCC 25922) around the meropenem disk were considered carbapenemase producer15.
Data and laboratory quality control
All quality control checks were done before, during, and after data collection. The questionnaire was pre-tested before the actual study was started. Standard Operating Procedures (SOP) were strictly followed for each procedure. The swab sample was transported and processed immediately after collection. If there is a delay in processing the specimen, the specimen is placed in the refrigerator. Before using the media, reagents, and antibiotic disks, the expiration dates were checked. Following sterility testing, the culture media was visually evaluated for cracks and thickness, as well as the presence of freezing, bubbles, and contaminants. Quality control was assured by concurrent testing with the American Type Culture Collection (ATCC) strains including E. coli ATCC 25,922, P. aeruginosa ATCC 27,852, and K. pneumoniae (ATCC 700603). For the ESBLs confirmatory test, ESBLs positive K. pneumoniae ATCC 700,603 and ESBLs negative E. coli ATCC 25,922 control strain was used. K. pneumoniae ATCC BAA-1705 was used as a positive control and K. pneumoniae ATCC BAA-1706 was used as a negative control for carbapenemase detection.
Data analysis and interpretation
The data were entered into Epi Data version 3.1 and exported to Statistical Package for Social Sciences (SPSS) version 25 for analysis. Descriptive statistics (median, percentages, or frequency) and logistic regression were computed. The bivariate logistic regression analysis was used to observe the relationship between the dependent variable and independent variables. Variables that showed P-value ≤ 0.2 bivariate logistic regression analyses were selected for further analysis using the multivariable logistic regression model. Any variables that showed a p-value ≤ 0.05 by multivariable logistic regression model were considered as statistically significant. Finally, the results were presented in words, graphs, and tables.
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