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Pseudomonas aeruginosa (PSA) and Acinetobacter baumannii (ACB) are non-fermentative bacteria mostly associated with nosocomial infections in humans. This study aimed to determine the antimicrobial resistance profiles and virulence gene of PSA and ACB previously isolated from humans in selected health facilities in Yaoundé, Cameroon. A total of 77 and 27 presumptive PSA and ACB isolates, respectively, were collected from the Yaoundé teaching hospital. These isolates were previously isolated from various samples including pus, blood and broncho-alveolar lavage. The identities of the isolates were determined through polymerase chain reaction (PCR) amplification of PSA and ACB specific sequences. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer disc diffusion method. Phenotypical expression of AmpC β-lactamases (AmpC), extended spectrum β-lactamases (ESBLs) and metallo β-Lactamases (MBLs) were determined using the combined disc method. Bacterial genomes were screened for the presence of β-lactamases bla_TEM and bla_CTXM genes using specific PCR. The pathogenicity of PSA and ACB was assessed through amplification of the lasB, exoA, pslA and exoS as well as OmpA and csuE virulence genes, respectively. Of the 77 presumptive PSA isolates, a large proportion (75 to 97.4%) were positively identified. All (100%) of the presumptive 27 ACB harbored the ACB-specific ITS gene fragment by PCR. Twenty five percent of the PSA isolates produced ESBLs phenotypically while more than 90% of these isolates were positive for the lasB, exoA …