Abstract
BackgroundAquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages.ResultsHere we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls.ConclusionsThese observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0281-8) contains supplementary material, which is available to authorized users.
Highlights
Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions
Quantitative polymerase chain reaction data have shown that Cryptosporidium can multiply within aquatic biofilms, with a 2–3 fold increase in Cryptosporidium numbers observed over a 6 day period [19]
To gain more detailed information about the dynamics and developmental biology of Cryptosporidium after exposure to biofilms, we have examined stages of Cryptosporidium produced in biofilms using confocal microscopy and scanning electron microscopy (SEM)
Summary
Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may support the growth of additional Cryptosporidium stages. Studies have shown that Cryptosporidium oocysts incorporate readily into biofilms [4,5,6], and concerns have been expressed over the sudden sloughing of biofilms from water pipes with an accumulated aggregation or ‘bolus’ of oocysts that. As Cryptosporidium was believed to be an obligate intracellular parasite and, lacking the capability to proliferate within biofilms [12], initial studies focused only on the association of the oocyst stage within biofilms [5,6,7,9]. Quantitative polymerase chain reaction (qPCR) data have shown that Cryptosporidium can multiply within aquatic biofilms, with a 2–3 fold increase in Cryptosporidium numbers observed over a 6 day period [19]. Because excysted oocysts have a distinctly different morphology from that of intact oocysts and can be distinguished readily by flow cytometry [20,21,22], we have used flow cytometry to track morphological changes to oocysts after exposure to an artificial biofilm system
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