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Objectives: Bloodstream infection (BSI) causes significant morbidity and mortality in patients, especially in developing countries. Changes in the epidemiological pattern of microorganisms, as well as the growth of antibacterial resistance have become important health concerns in Iran. The aims of this study were to evaluate the spectrum of pathogens causing BSIs in hospitalized patients in Shiraz, Iran, and their corresponding antimicrobial resistance patterns. Materials and Methods: In this retrospective study, 1585 positive blood samples were analyzed from March 2013 to March 2014. Samples from all hospitals within Shiraz were transferred to Professor Alborzi Clinical Microbiology Center. Then, the isolates were identified according to standard methods such as the analytical profile index system, and antibiotic susceptibility patterns were established consistent with the recommendations of the Clinical and Laboratory Standards Institute. Results: Coagulase-negative staphylococci (39%), Staphylococcus aureus (15.3%), Escherichia coli (8.5%), Pseudomonas spp. (7.5%), Enterococcus spp. (7.3%), and Acinetobacter spp. (6.6%) were the most frequent bacteria isolated from blood cultures. Linezolid and vancomycin (VA) had the highest effectiveness against gram-positive bacteria and gram-negative bacteria had high sensitivity to polymyxin B and colistin. Totally, 56.2% of Enterococcus isolates were VA-resistant (VRE) and 55.2% (122) of S. aureus were methicillin-resistant (MRSA). Finally, 59.02% (72) of E. coli isolates, 33.3% (5) of Serratia spp., and 42.85% (33) of Klebsiella spp. were extended-spectrum β-lactamase (ESBL) positive. Conclusions: Considering the results, the emergence of resistant strains such as MRSA, VRE, and ESBL is an alarming threat that would be a severe clinical issue with critical restrictions on antibiotic therapy. [ABSTRACT FROM AUTHOR]