AbstractBackground and AimsResistance to antibiotics and the capability to develop biofilm as two main virulent determinants ofKlebsiella pneumoniaehave important role in infection persistence. The aim of the study was to evaluate the association between the prevalence of aminoglycoside resistance and virulence genes and biofilm formation capacity inK. pneumoniaestrains isolated from hospitalized patients in South‐West of Iran.MethodsA total of 114 non‐duplicate clinical isolates ofK. pneumoniaecollected from Ahvaz teaching hospitals. Identification of species was performed by biochemical tests and then confirmed by polymerase chain reaction (PCR) ofrpoBgene. The susceptibility to antibiotics was determined by Kirby−Bauer disk diffusion method. Biofilm formation was assessed by microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants including fimbrial genes, aminoglycoside modifying enzymes‐ and 16S rRNA methylase (RMTase) genes.ResultsTotally, all collected strains were carbapenem resistant and showed multidrug‐ and extensively drug‐resistance phenotype (75% and 25%, respectively). Seventy‐one percent (n= 81) of isolates were non‐susceptible to aminoglycosides. Among aminoglycoside antibiotics,K. pneumoniaeisolates showed the highest and lowest resistance rates to tobramycin (71%) and the amikacin (25%), respectively. All biofilm producer strains were positive for the presence virulence determinants includingecpA, fimA, mrkD, andmrkA. Of 81 aminoglycosides non‐susceptible isolates 33% were positive for the presenceant (2″)‐Iaas the most prevalent gene followed byaac (3′)‐IIaandarmA(27%),aac (6′)‐Ib(18%), andaph (3′)‐Ia(15%).ConclusionK. pneumoniaeisolates showed the highest and the lowest aminoglycoside resistance rates to tobramycin and amikacin, respectively. Majority of isolates were biofilm producers and there was significant association between antibiotic resistance pattern and the strength of biofilm production. Theant(2″)‐Ia, aac (3′)‐IIa, andarmAgenes in aminoglycoside‐resistant isolates.