Municipal wastewater treatment plants as removal systems and environmental sources of human-virulent microsporidian spores

Abstract

Municipal wastewater treatment plants play a vital role in reducing the microbial load of sewage before the end-products are discharged to surface waters (final effluent) or local environments (biosolids). This study was to investigate the presence of human-virulent microsporidian spores (Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon hellem) and enterococci during treatment processes at four Irish municipal secondary wastewater treatment plants (plants A-D). Microsporidian abundance was significantly related to seasonal increase in water temperature. Plant A had the least efficient removal of E. intestinalis spores (32%) in wastewater, with almost 100% removal at other plants both in April and July. Some negative removal efficiencies were obtained for E. bieneusi (at plants C and D, -100%) and for E. hellem (at plants A and D, -90% and -50%). In addition, a positive correlation was found between the levels of enterococci and E. bieneusi in July (r (s) = 0.72, P < 0.05). In terms of the dewatered biosolids, a median concentration as high as 32,000 spores/Kg of E. hellem was observed at plant D in July. Plant C sewage sludge contained the lowest microsporidian loadings (E. bieneusi; 450 spores/L and 1,000 spores/L in April and July, respectively). This study highlights the seasonal variation in concentrations of microsporidian spores in the incoming sewage. Spores in final effluents and dewatered biosolids can be the source of human-virulent microsporidian contamination to the local environment. This emphasizes a considerably high public health risk when sewage-derived biosolids are spread during summer months. This study also suggested enterococci as a potential indicator of the presence of microsporidian spores in wastewater, especially for E. bieneusi.

Microsporidia

“…The liquid sewage sludge samples from one WWTP contained the lowest microsporidian loadings with E. bieneusi concentrations of 450 spores/L in April and 1,000 spores/L in July, and E. hellem concentration of 400 spores/L in April and zero in July. E. intestinalis was not identified in these sludge samples (Cheng et al, 2011). In Tunisia, 12 sludge samples were collected from 5 WWTPs, and E. bieneusi was identified by PCR in one dry sludge sample and seven dehydrated sludge samples (Ben .…”

“…The general scheme of these procedures utilizes preenrichment filtration followed by immunomagnetic bead separation (IMS) assay and detection by immunofluorescence antibody staining (FA) (Didier et al, 2004). Variations on this approach for detecting microsporidia in water samples include application of the IMS followed by PCR (Dowd et al, 1999;Sorel et al, 2003), water filtration followed by PCR (Sparfel et al, 1997), calcium carbonate flocculation followed by PCR (Hu et al, 2014), concentration of microsporidia by continuous separation channel centrifugation (Borchardt and Spencer, 2002), and sedimentation followed by fluorescent in situ hybridization (FISH) using species-specific fluorochromelabelled probes (Cheng et al, 2011;Lucy et al, 2008).…”

“…Thus further processing is still required to treat these products for inactivation and disintegration of microsporidian spores, such as ultrasonic treatment and quicklime stabilization. In addition, a significant correlation was found between the levels of enterococci and humanpathogenic E. bieneusi spores during the wastewater treatment processes, suggesting that enterococci may have potential as an indicator for the possible presence of human-pathogenic microsporidian spores in wastewater (Cheng et al, 2011).…”

“…The average concentration of E. hellem spores in wetland effluents of 29 ± 13.3 spores/L was significantly higher than that of E. bieneusi (7 ± 1.9 spores/L) in effluents . Also in northwestern Ireland, wastewater samples from four WWTPs were examined by multiplex FISH once in April and once in July, and E. bieneusi, E. hellem, and E. intestinalis spores were detected in effluent water from 3, 3, and 1 WWTP, respectively (Cheng et al, 2011). One study in the United States has also estimated the presence of humanpathogenic microsporidia in environmental samples collected from livestock production facilities in eastern Nebraska.…”

“…Since 2000, there has been an increase both in the overall percentage and volumes of sewage sludges spread on Irish farmland, with an estimated 45,543 tonnes spread in 2005 and 86,411 tonnes in 2007 (Cheng et al, 2011;Lucy et al, 2008). These large volumes of spread biosolids increase the risk of pathogens like microsporidia spores being introduced via surface runoff into waters used for drinking-water (Lucy et al, 2008).…”