Development of multiplex PCR assays for detecting Chlamydia-related bacteria and their natural host, free-living amoebae

In addition to Chlamydiaceae, eight novel families have been discovered to belong to the phylum Chlamydiae. The eight families are Parachlamydiaceae, Waddliaceae, Criblamydiaceae, Parilichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, Clavichlamydiaceae and Piscichlamydiaceae.These families are phylogenetic relatives to Chlamydiaceae, share the intracellular developmental cycle and are widely distributed in nature and are therefore referred to as environmental Chlamydiae or Chlamydia related bacteria (CRB). CRB have a broad range of potential hosts. All families except Criblamydiaceae cause disease in animals and infect for example fish, arthropods and cattle. Families Parachlamydiaceae, Waddliaceae, Rhabdochlamydiaceae and Simkaniaceae are also shown to cause respiratory disease and adverse pregnancy outcomes in human. Free-living amoebae (FLA) are natural hosts of some CRB. CRB are able to survive and replicate inside of FLA that offers protection and nutrients for CRB. It has been suggested that CRB are transported to new environments inside of FLA. CRB DNA has previously been found on human skin (Hokynar et al. 2016, Hokynar et al. 2018, Tolkki et al. 2018) and in our water distribution system. CRB distributed to our tap- and shower water systems inside of FLA (Thomas and Ashbolt 2011) could be a potential rout of transmission of CRB DNA to human skin. As the diversity and size of the CRB group is large and CRB are very laborious to grow in vitro, it is challenging to detect CRB and to study their pathogenicity. Detection of CRB in clinical and environmental samples is mainly based on PCR methods. A non species-specific PCR method targeting Chlamydiales 16S rRNA (PanChl16S), that in theory amplifies all known CRB, has successfully been used in detection, but post PCR sequencing of the amplicon is required to identify the species. Also, more specific quantitative PCRs have been designed to detect specific families or species of Chlamydiae. However, the volume of clinical specimens available is often limited and allows only few separate analyzes. Due to the challenges identified with detection of CRB, efficient multiplex PCR assays would save time and resources and would be useful tools when detecting CRB DNA. The objective of the work was to explore the possibility of applying multiplexed analyzes to a limited specimen volume effectively. One aim of this thesis was to set up two multiplex PCR assays for detection of seven different CRB and a multiplex PCR for detection of three different FLA. Another aim of the work was to analyze the possibility of CRB to be transported to human skin from our water distribution system inside of FLA. In this thesis we set up two multiplex PCR assays for detection of CRB reference strains P. acanthamoebae, C. sequanensis, S. negevensis, Protochlamydia spp., Rhabdochlamydia spp., W. chondrophila and E. lausannensis. We also set up two PCR assays for detection of three different FLA reference strains: Acanthamoeba spp., Vahlkampfiidae spp., and V. vermiformis. We succeeded in developing two real-time multiplex PCR assays for detection of CRB DNA and two real-time PCR assay for detection of FLA DNA. Variability between replicates for each PCR target was low and the detection limit (100%) for each target ranged from 50-500 control plasmid copies per PCR reaction. The R2-value for each target was ≥0.98 and the reaction efficiency for each target ranged from 82-111%. Samples collected from showerheads (n=18) and water filters (n=2) as well as skin swabs (n=27) were studied with these newly established assays and PanChl16S PCR. The results obtained with the multiplex assays developed in this study were similar to the results obtained with the PanChl16S. CRB DNA was detected in 67% of the showerhead samples, in 100% of the water filter samples and in 31% of the skin swabs. Amoebae DNA was detected in 80% of the showerhead samples. Our results confirm earlier observation that Chlamydiae DNA is frequently observed in human skin swabs and suggest that CRB could be transported to human skin from our water distribution system inside of FLA.