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Background: As part of the containment of the COVID-19 pandemic, mobile handwashing stations (mHWS) were deployed in healthcare facilities in low-resource settings. We assessed mHWS in hospitals in the Democratic Republic of the Congo for contamination with Gram-negative bacteria., Methods: Water and soap samples of in-use mHWS in hospitals in Kinshasa and Lubumbashi were quantitatively cultured for Gram-negative bacteria which were tested for antibiotic susceptibility. Meropenem resistant isolates were assessed for carbapenemase enzymes using inhibitor-based disk and immunochromatographic tests. Mobile handwashing stations that grew Gram-negative bacteria at counts > 10,000 colony forming units/ml from water or soap were defined as highly contaminated., Results: In 26 hospitals, 281 mHWS were sampled; 92.5% had the "bucket with hand-operated tap" design, 50.5% had soap available. Overall, 70.5% of mHWS grew Gram-negative bacteria; 35.2% (in 21/26 hospitals) were highly contaminated. Isolates from water samples (n = 420) comprised 50.3% Enterobacterales (Klebsiella spp., Citrobacter freundii, Enterobacter cloacae), 14.8% Pseudomonas aeruginosa and 35.0% other non-fermentative Gram-negative bacteria (NFGNB, including Chromobacterium violaceum and Acinetobacter baumannii). Isolates from soap samples (n = 56) comprised Enterobacterales (67.9%, including Pluralibacter gergoviae (n = 13)); P. aeruginosa (n = 12) and other NFGNB (n = 6). Nearly one-third (31.2%, 73/234) of Enterobacterales (water and soap isolates combined) were multi-drug resistant; 13 isolates (5.5%) were meropenem-resistant including 10 New Delhi metallo-beta-lactamase (NDM) producers. Among P. aeruginosa and the other NFGNB, 7/198 (3.5%) isolates were meropenem resistant, 2 were NDM producers. Bacteria listed as critical or high priority on the World Health Organization Bacterial Priority Pathogens List accounted for 20.3% of isolates and were present in 12.0% of all mHWS across 13/26 hospitals. Half (50.5%) of highly contaminated mHWS were used by healthcare workers and patients as well as by caretakers and visitors., Conclusions: More than one third of in-use mobile handwash stations in healthcare facilities in a low resource setting were highly contaminated with clinically relevant bacteria, part of which were multidrug resistant. The findings urge a rethink of the place of mobile handwash stations in healthcare facilities and to consider measures to prevent their contamination., Competing Interests: Declarations. Ethics approval and consent to participate: The study was approved by the Institutional Review Board of the Institute of Tropical Medicine, Antwerp, Belgium (1434/20) and the ethics committee of the School of Public Health of the University of Kinshasa, DRC (ESP/CE/208/2020). Written support for the study was granted by the General Health Office of the DRC Ministry of Health. All hospital directors and interviewees provided consent. Results presented were aggregated for all hospitals and no results of individual hospitals were presented. Competing interests: The authors declare no competing interests. Consent for publication: Not Applicable., (© 2024. The Author(s).)