BackgroundAntimicrobial resistance (AMR) has emerged as a major global public health challenge due to the overuse and misuse of antibiotics for humans and animals. Hospitals are among the major users of antibiotics, thereby having a large contribution to AMR.ObjectiveThe aim of this study is to determine the prevalence of antibiotic-resistant pathogenic bacteria and the level of antibiotic residues in the hospital effluents in Selangor, Malaysia.MethodsA cross-sectional study will be performed in the state of Selangor, Malaysia. Tertiary hospitals will be identified based on the inclusion and exclusion criteria. The methods are divided into three phases: sample collection, microbiological analysis, and chemical analysis. Microbiological analyses will include the isolation of bacteria from hospital effluents by culturing on selective media. Antibiotic sensitivity testing will be performed on the isolated bacteria against ceftriaxone, ciprofloxacin, meropenem, vancomycin, colistin, and piperacillin/tazobactam. The identification of bacteria will be confirmed using 16S RNA polymerase chain reaction (PCR) and multiplex PCR will be performed to detect resistance genes (ermB,mecA,blaNDM-L,blaCTX-M,blaOXA-48,blaSHV,VanA,VanB,VanC1,mcr-1,mcr-2,mcr-3,Intl1,Intl2,andqnrA). Finally, the level of antibiotic residues will be measured using ultrahigh-performance liquid chromatography.ResultsThe expected outcomes will be the prevalence of antibiotic-resistantEnterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa, andEnterobacter(ESKAPE) bacterial species from the hospital effluents, the occurrence of antibiotic resistance genes (ARGs) from the isolated ESKAPE bacteria, and the level of antibiotic residues that may be detected from the effluent. Sampling has been conducted in three hospitals. Data analysis from one hospital showed that as of July 2022, 80% (8/10) ofE. faeciumisolates were resistant to vancomycin and 10% (1/10) were resistant to ciprofloxacin. Further analysis will be conducted to determine if the isolates harbor any ARGs and effluent samples are being analyzed to detect antibiotic residues. Sampling activities will be resumed after being suspended due to the COVID-19 pandemic and are scheduled to end by December 2022.ConclusionsThis study will provide the first baseline information to elucidate the current status of AMR of highly pathogenic bacteria present in hospital effluents in Malaysia.International Registered Report Identifier (IRRID)DERR1-10.2196/39022