Capture and Retention of Cryptosporidium parvum Oocysts by Pseudomonas aeruginosa Biofilms

Abstract

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.The human pathogen Cryptosporidium parvum is responsible for numerous waterborne disease outbreaks in the United States (15,20,34,41,43). Outside its host, C. parvum exists as a nonreproductive oocyst, ϳ5 m in diameter, that is resistant to typical environmental stresses (6,29,39,40). C. parvum oocysts originate from the waste of infected hosts and are discharged in large quantities from municipal wastewater treatment facilities, animal agriculture, and wildlife populations (2,18,26,47). Because oocysts are persistent in the environment, the transmission of viable oocysts from sources to public water supplies can result in human infection even over long transport distances. Therefore, protection of public health requires a clear understanding of the factors that control the migration of Cryptosporidium in the environment.The transport of C. parvum oocysts can be influenced by interactions with surface-attached microbial communities, generally termed biofilms. Biofilms are ubiquitous in aquatic environments, where they form on rocks, plants, and sediments, and are also prevalent in wastewater treatment systems. Biofilm microorganisms are encased in a heterogeneous matrix of extracellular polymeric substances (EPS) composed of polysaccharides, proteins, lipids, and nucleic acids (10, 16). Both the morphology and chemical characteristics of biofilms are expected to promote the deposition and retention of C. parvum oocysts. Previous studies in both laboratory and environmental systems have shown that colloidal particles such as latex beads, bacteria, and virions can be readily transferred to biofilm communities from the surrounding bulk fluid (4,13,17,32,33,37,44,45). As...

Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory

“…In the present study, summer biofilms were the only biofilms that were significantly different in thickness, but their thicker biomass did not yield an increase in the number of oocysts attached to the biofilm (Table 1). Although not significant, a trend of increasing oocyst attachment with increasing surface roughness (measured as the standard deviation of biofilm thickness measurements) was observed, as seen by others using 1-m microbeads (5) and C. parvum oocysts (23). In addition, the experimental conditions (specifically, experimental temperature) did not appear to impact oocyst attachment to the biofilms either (Fig.…”

“…The quick steady-state deposition of particles has been found to be independent of cell density in the biofilm or biofilm thickness, but dependent on the particle surface properties (i.e., surface charge and hydrophobic properties) and the physical properties of the biofilm (i.e., surface roughness and pore size) (12,23). Accordingly, biofilm thickness did not appear to play a role in how many oocysts attached to the biofilms in the present study, as also shown by others (5,12,16,17).…”

“…Searcy et al (23) noted that, along with biofilm architecture, surface-chemical interactions and background water chemistry affect pathogen deposition in biofilms, as reflected by the significant difference in the fraction of C. parvum oocysts that attached to the biofilm using two different growth medias (LB broth and Jensen medium). Experiments performed by Searcy et al (23) showed that changing the growth medium impacts the surface charge of oocysts and therefore affects their attachment to a pure culture biofilm. This change in flow medium chemistry could be analogous to water chemistry changes with season; however, we did not see any significant changes across seasons in the bulk water chemistry in the present study (Fig.…”

“…Rogers and Keevil (22) showed that oocysts attached to a biofilm composed of a natural microbial assemblage collected from a reservoir at a concentration of 1,400 oocysts/cm 2 after the addition of 10 8 oocysts in 10 ml of sterile water. Dai and Hozalski (3) and Searcy et al (23) used pure culture biofilms to demonstrate oocyst attachment; however, only Searcy et al (23) accounted for sloughing, although no oocyst release from the biofilm was seen during the course of their experiments.…”

“…Several studies have examined pathogen transport dynamics in biofilms using glass or latex beads of various sizes as surrogates for pathogens (5,8,16,17). A few studies examined the attachment of C. parvum oocysts to biofilms but did not use natural microbial assemblages to make the biofilms (3,23) or quantify how many oocysts attached or sloughed (9,22). Rogers and Keevil (22) showed that oocysts attached to a biofilm composed of a natural microbial assemblage collected from a reservoir at a concentration of 1,400 oocysts/cm 2 after the addition of 10 8 oocysts in 10 ml of sterile water.…”