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As very little information is known of the antibiotic resistance in Gram-negative bacteria in Libya in addition to the desperate need for insight knowledge of the antibiotic resistance in Libyan hospitals, this study was undertaken to investigate the mechanism of antibiotic resistance in isolates collected from clinical, non-clinical and environmental samples from Tripoli and Benghazi, Libya. Bacterial collection include samples taken from patients admitted to the hospitals in ICUs and other wards, they also include swabs randomly collected from hospitals environment. This study clearly demonstrates the emergence of MDR Gram-negative bacteria in Tripoli and Benghazi hospitals, these MDR bacteria were clinical and non-clinical revealing the long standing infection control problem in these hospitals. Perhaps the most interesting finding of this study is blaTMB-1 which was detected in environmental strain swabbed from the floor of Tripoli central hospital. This MBL was unusual in terms of the similarity this gene shares with other known MBLs and also to the discovery of this MBL carried by environmental bacteria A. xylosoxidans, it is moreover the first MBL discovered in Libya.