According to Hefner's death certificate, obtained Tuesday by The Blast, the mogul contracted septicemia and an E. coli strain deemed "highly resistant to antibiotics" six days before his death. Septicemia is a blood infection that can be caused by E. coli.
The Los Angeles Department of Public Health was not able to determine how Hefner was initially infected with the bacteria, Daily Mail reported.
The business mogul was interred at the Los Angeles Westwood Village Memorial Park Cemetery next to Marilyn Monroe in a private ceremony.
Hefner's third wife, Crystal Harris, made her first statement Monday.
"I am heartbroken. I am still in disbelief," Harris told People. "He gave me life. He gave me direction. He taught me kindness. I will feel eternally grateful to have been by his side, holding his hand, and telling him how much I love him."
"He changed my life, he saved my life. He made me feel loved every single day," Harris concluded.
Hefner is survived by Harris and his four children from two previous marriages.