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Background: Staphylococcus aureus is an important cause of infection, and brings additional concern with methicillin resistance. In addition, nasal methicillin-resistant Staphylococcus aureus (MRSA) colonization rates among health care workers are higher than that for general population. To determine the prevalence rate and risk factors for the colonization of S. aureus, including MRSA, among janitors working in hospitals in northern Taiwan, we conducted this study. Methods: Between June and August, 2014, a total of 186 janitors, 111 working in hospitals and 75 working in non-medical institutions, were recruited. Specimens were obtained from the nares of the subjects for the detection of S. aureus, with a questionnaire completed for each subject. All the S. aureus isolates, including MRSA and methicillin-susceptible S. aureus (MSSA), were further molecularly characterized. Results: The nasal carriage rate of S. aureus was 15.3% for hospital janitors and 13.3% for non-medical janitors. The carriage rate of MRSA was 3.6% for hospital janitors and 1.3% for non-medical janitors. No statistically significant difference was found in the nasal carriage rate of S. aureus (p = 0.707) and MRSA (p = 0.65) between hospital janitors and non-medical janitors. Hospital janitors working in hospital more than 6 years and cleaning microbiologic laboratories were significantly associated with nasal S. aureus colonization. All 5 MRSA isolates carried either staphylococcal cassette chromosome type IV or V and three of them belonged to sequence type (ST) 59, the community clone prevailing in Taiwan. Of the 22 MSSA isolates, six pulsotypes were identified, with one major type for 14 isolates (shared by five STs) and another type for 4 isolates (all belonged to ST 188). Conclusion: Exposure to the hospital environment may not increase the nasal carriage rate of S. aureus, including MRSA, among janitors in hospitals in Taiwan. However, for janitors in the hospital setting, working for more than six years in hospital and cleaning laboratories may be risks factors for carrying S. aureus. [ABSTRACT FROM AUTHOR]