Mimivirus-like Particles in Acanthamoebae from Sewage Sludge

al. Burden of disease caused by Strepto-coccus pneumoniae in children younger than 5 years: global estimates. al. Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection.rial load in children and adolescents with pneumococcal pneumonia and empyema. cation of pneumococcal serotypes from culture-negative clinical specimens by novel real-time PCR. To the Editor: Mimivirus is a giant, double-stranded DNA virus. Its 650-nm diameter and 1.2-Mb genome make it the largest known virus (1). In 2003, mimivirus was isolated from a water cooling tower in Bradford, UK, after a pneumonia outbreak and was reported to infect Acanthamoeba polyphaga amebae (2). Subsequently, a small number of additional isolates have been reported (3). Mimivirus has been associated with pneumonia, and this association was strengthened after antibodies to mimivirus were found in serum samples from patients with community-and hospital-acquired pneumonia and after mimivirus DNA was found in bronchoalveolar lavage specimens (4). More direct evidence of pathogenicity was illustrated when a pneumonia-like disease developed in a laboratory technician who worked with mimivirus and showed seroconversion to 23 mimivirus-specifi c proteins (5). We report fi nding mimivirus-like particles during our molecular study of Acanthamoeba spp. abundance and diversity in fi nal-stage conventionally treated sewage sludge from a wastewater treatment plant in the West Midlands, UK. Using metagenomic DNA extracted from the sludge (6), we estimated the abundance of Acanthamoeba spp. by using real-time PCR (7) and found it to be ≈1 × 10 2 /g sludge. To assess species diversity, we amplifi ed an Acanthamoeba spp.–specifi c 18S rRNA target, which resulted in products of ≈450 bp (8). PCR products were cloned and sequenced, revealing low Acanthamoeba spp. diversity with a predominance of clones most similar to A. palestinensis (22/25 clones), which fall within the T6 clade according to the classifi cation of Stothard et al. (9). A small number (3/25) of clones showed closest similarity to acanthamoebae belonging to the T4 clade, which includes strains considered to be human pathogens, including some A. polyphaga strains. Acanthamoebae were isolated from fully digested sewage sludge by inoculating diluted sludge onto cerophyl-Prescott infusion agar and subculturing onto nonnutrient agar plates streaked with heat-killed Escherichia coli. Cultures were incubated at 20°C and 30°C and examined under an Axioskop 2 microscope (Zeiss, Oberkochen, Germany) at 100× magnifi cation; cells of interest were examined at 1,000× magnifi cation. One clonal population of an Acanthamoeba sp. isolated at …