Nested Species Distribution Models of Chlamydiales in Ixodes ricinus (Tick) Hosts in Switzerland.

The tick Ixodes ricinus is the vector of various pathogens, including Chlamydiales bacteria, which potentially cause respiratory infections. In this study, we modeled the spatial distribution of I. ricinus and associated Chlamydiales over Switzerland from 2009 to 2019. We used a total of 2,293 ticks and 186 Chlamydiales occurrences provided by a Swiss Army field campaign, a collaborative smartphone application, and a prospective campaign. For each tick location, we retrieved from Swiss federal data sets the environmental factors reflecting the topography, climate, and land cover. We then used the Maxent modeling technique to estimate the suitability of particular areas for I. ricinus and to subsequently build the nested niche of Chlamydiales bacteria. Results indicate that I. ricinus habitat suitability is determined by higher temperature and normalized difference vegetation index (NDVI) values, lower temperature during the driest months, and a higher percentage of artificial and forest areas. The performance of the model was improved when extracting the environmental variables for a 100-m radius buffer around the sampling points and when considering the climatic conditions of the 2 years previous to the sampling date. Chlamydiales bacteria were favored by a lower percentage of artificial surfaces, drier conditions, high precipitation during the coldest months, and short distances to wetlands. From 2009 to 2018, we observed an extension of areas suitable to ticks and Chlamydiales, associated with a shift toward higher altitude. The importance of considering spatiotemporal variations in the environmental conditions for obtaining better prediction was also demonstrated.IMPORTANCEIxodes ricinus is the vector of pathogens including the agent of Lyme disease, the tick-borne encephalitis virus, and the less well-known Chlamydiales bacteria, which are responsible for certain respiratory infections. In this study, we identified the environmental factors influencing the presence of I. ricinus and Chlamydiales in Switzerland and generated maps of their distribution from 2009 to 2018. We found an important expansion of suitable areas for both the tick and the bacteria during the last decade. Results also provided the environmental factors that determine the presence of Chlamydiales within ticks. Distribution maps as generated here are expected to bring valuable information for decision makers in controlling tick-borne diseases in Switzerland and establishing prevention campaigns. The methodological framework presented could be used to predict the distribution and spread of other host-pathogen pairs to identify environmental factors driving their distribution and to develop control or prevention strategies accordingly.

Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Marine Sediments Illuminate Chlamydiae Diversity and Evolution.

The bacterial phylum Chlamydiae is so far composed of obligate symbionts of eukaryotic hosts. Well known for Chlamydiaceae, pathogens of humans and other animals, Chlamydiae also include so-called environmental lineages that primarily infect microbial eukaryotes. Environmental surveys indicate that Chlamydiae are found in a wider range of environments than anticipated previously. However, the vast majority of this chlamydial diversity has been underexplored, biasing our current understanding of their biology, ecological importance, and evolution. Here, we report that previously undetected and active chlamydial lineages dominate microbial communities in deep anoxic marine sediments taken from the Arctic Mid-Ocean Ridge. Reaching relative abundances of up to 43% of the bacterial community, and a maximum diversity of 163 different species-level taxonomic units, these Chlamydiae represent important community members. Using genome-resolved metagenomics, we reconstructed 24 draft chlamydial genomes, expanding by over a third the known genomic diversity in this phylum. Phylogenomic analyses revealed several novel clades across the phylum, including a previously unknown sister lineage of the Chlamydiaceae, providing new insights into the origin of pathogenicity in this family. We were unable to identify putative eukaryotic hosts for these marine sediment chlamydiae, despite identifying genomic features that may be indicative of host-association. The high abundance and genomic diversity of Chlamydiae in these anoxic marine sediments indicate that some members could play an important, and thus far overlooked, ecological role in such environments and may indicate alternate lifestyle strategies.

Apoptosis Functions in Defense against Infection of Mammalian Cells with Environmental Chlamydiae.

Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All Chlamydiales are obligate intracellular bacteria, and different species infect vastly different hosts. Chlamydia trachomatis infects human epithelial cells; Parachlamydia acanthamoebae replicates in amoebae. We here report that apoptosis impedes growth of P. acanthamoebae in mammalian cells. In HeLa human epithelial cells, P. acanthamoebae infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-XL or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of P. acanthamoebae in HeLa cells, as measured by fluorescence in situ hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with C. trachomatis inhibited P. acanthamoebae-induced apoptosis, suggesting that the known antiapoptotic activity of C. trachomatis can also block P. acanthamoebae-induced apoptosis. C. trachomatis coinfection could not rescue P. acanthamoebae growth in HeLa; in coinfected cells, C. trachomatis even suppressed the growth of P. acanthamoebae independently of apoptosis, while P. acanthamoebae surprisingly enhanced the growth of C. trachomatis Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.

Simkania negevensis in Crohn's Disease.

BACKGROUND: Simkania negevensis is an obligate intracellular Gram-negative bacterium (family Simkaniaceae, order Chlamydiales) that has been isolated from domestic and mains water supplies, is able to infect human macrophages, and can induce an inflammatory response in the host. METHODS: From June to December 2016, in a single-center observational study, colonic Crohn's disease patients and controls (subjects undergoing screening for colorectal cancer) underwent blood tests to identify serum-specific immunoglobulin G (IgG) and immunoglobulin A (IgA) to S. negevensis and a colonoscopy with biopsies for detection of S. negevensis DNA by polymerase chain reaction (PCR). RESULTS: Forty-three Crohn's disease patients and 18 controls were enrolled. Crohn's disease patients had higher prevalence of IgA antibodies to S. negevensis compared with controls (20.9% versus 0%, p = 0.04). Simkaniaceae negevensis DNA was detected in 34.9% and 5.6% of intestinal biopsies in Crohn's disease patients and controls, respectively (p = 0.02). All Crohn's disease patients with PCR-positive biopsies for S. negevensis were IgG seropositive, with specific IgA in 60% of them (p < 0.001). Immunosuppressive therapies, extraintestinal manifestations, or disease activity did not influence the presence of S. negevensis in the Crohn's disease population. CONCLUSIONS: We identified S. negevensis in Crohn's disease patients by demonstrating the presence of S. negevensis mucosal DNA and seropositivity to the bacterium. These results could support the presence of an acute or persistent S. negevensis infection and suggest a possible role in the pathogenesis of Crohn's disease.

The first detection of the sequence of bacteria from the Simkaniaceae family in surface waters in Poland.

One of the most common reasons for horse lameness is subchondral bone cysts (SBCs), which are especially evident in young horse athletes. It is believed that SBC development is strongly associated with an individual's bone growth and/or bone microstructure impairment. Current methods of SBC treatment include pharmacological treatment or surgical procedures which may allow the bone within the cyst to rebuild and be restored to properly developed bone tissue. Thus, we propose filling the SBCs with a 3D complex of alginate hydrogel and autologous adipose derived mesenchymal stem cells (ASCs). We have observed at the in vitro level, that this hydrogel complex induces osteogenic and chondrogenic differentiation potential through the upregulation of bone morphogenetic protein, osteopontin, collagen type I and aggrecan mRNA levels. Moreover, we detected the creation of a 3D extracellular matrix (EM). To investigate the complex in vivo, we chose 8 horses of varying age suffering from SBC, which resulted in lameness, to undergo experimental surgery. We documented the horses' clinical appearance, lameness and radiographic appearance, to determine that there was clinical improvement in 87.75% of the patients (n=7, out of 8 horses) 6 months postoperatively and 100% (n=8, out of 8 horses) a year after surgery. These results are promising for the potential of this procedure to become the standard in SBC treatment.

Genotyping of Candidatus Syngnamydia salmonis (chlamydiales; Simkaniaceae) co-cultured in Paramoeba perurans (amoebozoa; Paramoebidae).

Candidatus Syngnamydia salmonis (Chlamydiales, Simkaniaceae) was described as an epitheliocystis-causing bacterium from the gills of Atlantic salmon (Salmo salar) in Norway. A bacterium showing 99.2% 16S rRNA identity to Cand. S. salmonis is able to multiply in Paramoeba perurans and based on the classification criteria this bacterium could represent the same species as Cand. S. salmonis. Sequencing the genome of the cultured bacterium has made it possible to fulfill the minimal standards for genetic characterization of species within the order Chlamydiales. The complete rRNA genes, the amino acid sequences of SucA, PepF, Adk, HemL, DnaA, FtsK and FabI, are presented in addition to the morphology of the Chlamydia-like morphs in the cytoplasm of P. perurans.

Presence of Waddlia chondrophila in hot water systems from non-domestic buildings in France.

The presence of Waddlia chondrophila has been related to respiratory tract infections and human and animal fetal death. Although several sources of infection have been suggested, the actual source remains unknown and limited information exists on the prevalence of W. chondrophila in the environment. This pathogen has been previously detected in well water but its presence has not been confirmed in water networks. Since these bacteria have been detected in water reservoirs, it has been hypothesized that they can access artificial water systems and survive until they find appropriate conditions to proliferate. In this work, their presence in water samples from 19 non-domestic water networks was tested by quantitative polymerase chain reaction (qPCR). Approximately half of the networks (47%) were positive for W. chondrophila and the overall results revealed 20% positive samples (12/59). Furthermore, most of the samples showed low concentrations of the pathogen (<200 genomic units/L). This finding demonstrates that W. chondrophila can colonize some water networks. Therefore, they must be considered as potential infection sources in future epidemiological studies.

Molecular evidence of Chlamydiales in ticks from wild and domestic hosts in Sardinia, Italy.

Ticks are well known to be important vectors for a wide range of bacteria, viruses and protozoa affecting human and animal health. Ixodid ticks are widely distributed in Sardinia, and an increasing number of tick-borne bacteria have been documented in the island. A growing number of evidence are supporting the hypothesis of alternative transmission routes for chlamydial bacteria such as the involvement of vectors. This study was conducted to provide possible molecular detection of members belonging to the Chlamydiales order in Sardinian ticks and to update information concerning the presence of new ectoparasite-borne bacteria in ticks collected from domestic and wild hosts in a typical Mediterranean environment. A total of 378 ticks were individually screened with a pan-Chlamydiales specific primers targeting the 16S rRNA gene. Chlamydiales DNA was detected in 28% of the total ticks analyzed. The analyses of sequences highlighted that Rhipicephalus sanguineus sensu lato, Rhipicephalus bursa, Rhipicephalus annulatus, Haemaphysalis sulcata, Haemaphysalis punctata and Dermacentor marginatus ticks exhibited DNA of Chlamydiaceae and Parachlamydiaceae members. Our results revealed that DNA of zoonotic microorganisms such as C. psittaci, C. abortus and the emerging pathogen Parachlamydia acanthamoebae are present in Sardinian ticks. Since routes of Chlamydia transmission are yet to be fully defined, the role of ticks as possible vectors for Chlamydiales remains the most challenging and interesting question to be addressed in future research. Continued monitoring of these pathogens in tick vectors is needed to provide strategies for controlling of possible chlamydial infections and disease outbreaks in the island.

Candidatus Actinochlamydia pangasiae sp. nov. (Chlamydiales, Actinochlamydiaceae), a bacterium associated with epitheliocystis in Pangasianodon hypophthalmus.

Chlamydial infections are recognised as causative agent of epitheliocystis, reported from over 90 fish species. In the present study, the farmed striped catfish Pangasianodon hypophthalmus (14-15 cm, 70-90 g) with a history of cumulative mortality of about 23% during June and July 2015, were brought to the laboratory. The histopathological examination of gills from the affected fish revealed presence of granular basophilic intracellular inclusions, mostly at the base of the interlamellar region and in gill filaments. A concurrent infection with Trichodina spp., Ichthyobodo spp. and Dactylogyrus spp. was observed in the gills. The presence of chlamydial DNA in the gills of affected fish was confirmed by amplification and sequencing of 16S rRNA gene. BLAST-n analysis of these amplicons revealed maximum similarity (96%) with Candidatus Actinochlamydia clariae. On the basis of phylogenetic analysis, it was inferred that the epitheliocystis agents from striped catfish were novel and belonged to the taxon Ca. Actinochlamydia. It is proposed that epitheliocystis agents from striped catfish will be named as Ca. Actinochlamydia pangasiae. The 16S rRNA gene amplicons from novel chlamydiae were labelled and linked to inclusions by in situ hybridisation. This is the first report of epitheliocystis from India in a new fish host P. hypophthalmus.

Molecular prevalence of Chlamydia and Chlamydia-like bacteria in Tunisian domestic ruminant farms and their influencing risk factors.

Chlamydia and Chlamydia-like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan-Chlamydiales real-time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population-level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd-level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central-eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.

Developmental Cycle and Genome Analysis of Protochlamydia massiliensis sp. nov. a New Species in the Parachlamydiacae Family.

Amoeba-associated microorganisms (AAMs) are frequently isolated from water networks. In this paper, we report the isolation and characterization of Protochlamydia massiliensis, an obligate intracellular Gram-negative bacterium belonging to the Parachlamydiaceae family in the Chlamydiales order, from a cooling water tower. This bacterium was isolated on Vermamoeba vermiformis. It has a multiple range of hosts among amoeba and is characterized by a typical replication cycle of Chlamydiae with a particularity, recently shown in some chlamydia, which is the absence of inclusion vacuoles in the V. vermiformis host, adding by this a new member of Chlamydiae undergoing developmental cycle changes in the newly adapted host V. vermiformis. Draft genome sequencing revealed a chromosome of 2.86 Mb consisting of four contigs and a plasmid of 92 Kb.

Molecular evidence for the absence of an association between Simkania negevensis and respiratory diseases.

Simkania negevensis has been implicated in respiratory diseases. This study aimed to unveil the aetiological role of this bacterium in community-acquired pneumonia (CAP) and bronchitis in Jordanian adults. Nasopharyngeal samples were collected from 98 CAP or bronchitis patients and 96 control individuals, and tested for Simkania nucleic acids using a PCR assay. The overall prevalence of the bacterial DNA in patients was markedly high and reached 57.1 %. Intriguingly, Simkania DNA was detected in 62.5 % of the nasopharyngeal swabs collected from apparently healthy controls (P>0.05). The DNA positivity in the bronchitis and CAP subgroups was 57.7 and 56.9 %, respectively, percentages that are approximately comparable to the DNA positivity assessed for the entire patient population. Simkania is most likely not a causative agent of CAP or bronchitis, despite its remarkable high prevalence. This organism, in the nasopharynx, is potentially harmless to the host and may coexist in a commensal relationship.

Seroprevalence of a "new" bacterium, Simkania negevensis, in renal transplant recipients and in hemodialysis patients.

BACKGROUND: Simkania negevensis is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydiales order. It is considered an ubiquitous microorganism and aquatic environments may be involved as a source of infection for humans. It was just isolated in samples from domestic water supplies and from mains water supplies, like spa water or swimming pool water, confirming its ability to resist to the common chlorination treatments. Evidence indicates a possible role of the microorganism in respiratory tract infections, in gastroenteric disorders and in the pathogenesis of cardiovascular disease, furthermore it has hypothesized that it could play a role in lung transplant rejection. Prevalence and possible effects in nephrology are unknown. METHODS: We examined the occurrence of Simkania negevensis in two differents populations, both characterized by a high susceptibility to infectious complications: 105 hemodialysis patients, 105 renal transplant recipients and 105 healthy subjects through the IgG and IgA response to Simkania negevensis in their sera. Serum antibodies to Simkania negevensis were detected by a homemade ELISA performed according to the Kahane's protocol. Furthermore water samples from hemodialytic circuit were collected, to evaluate Simkania negevensis resistance to usual treatment of disinfection. RESULTS: Our results were unexpected, showing a higher seroprevalence of antibodies against Simkania negevensis in the hemodialysis patients, compared to renal transplant patients (IgG 22% vs 9% - IgA 9% vs 3%). S. negevensis was isolated in all water samples analyzed. CONCLUSIONS: Our study detected for the first time the occurrence of S. negevensis in hemodialysis and in renal transplant patients. Our findings suggest that water used in hemodialysis could be one of the possible sources of S. negevensis infection, without clinical involvement risk for patients.

Chlamydia trachomatis and chlamydia-like bacteria: new enemies of human pregnancies.

PURPOSE OF REVIEW: This review provides an update on the roles of Chlamydia trachomatis and the related Waddlia chondrophila and Parachlamydia acanthamoebae in miscarriage, stillbirths and preterm labour in humans. A broad audience, including microbiologist, infectiologists, obstetricians and gynaecologists, should be aware of the potential threat of these Chlamydiales for human reproduction. RECENT FINDINGS: Despite increasing laboratory techniques and possibilities to perform diagnostic tests, the cause of miscarriage is only identified in 50% of the cases. Intracellular bacteria, such as C. trachomatis and Chlamydia-related bacteria, are difficult to detect in routine clinical samples and could represent possible agents of miscarriages. C. trachomatis is considered the world largest sexual transmitted bacterial agent and is associated with adverse pregnancy outcome in human. In the last decade Chlamydia-like organisms, such as W. chondrophila and P. acanthamoebae, have also been associated with adverse pregnancy outcomes in human and/or animals. SUMMARY: We review here the current evidences for a pathogenic role in humans, the diagnostic approaches and possible treatment options of C. trachomatis, W. chondrophila and P. acanthamoebae.

Protochlamydia phocaeensis sp. nov., a new Chlamydiales species with host dependent replication cycle.

Chlamydiae are pathogenic and symbiotic bacteria, which form an important part of amoeba-associated microorganisms. In this paper, we report the isolation, developmental cycle and genome analysis of Protochlamydia phocaeensis sp. nov., an obligate intracellular parasite with a large host spectrum, able to infect Acanthamoeba castellanii, Acanthamoeba polyphaga, and Vermamoeba vermiformis. The genome size is 3,424,182 bp with a GC content of 42%. This bacterium displayed a particular developmental cycle depending on the infected host. The P. phocaeensis showed typical inclusion vacuoles in A. castellanii, while these were absent in V. vermiformis. Since "Chlamydiae-amoebae" interactions are supposed to depend on the chlamydial species, our findings speculate that variations in the developmental cycle of certain Chlamydiae are also host dependent.

Simkania negevensis, an insight into the biology and clinical importance of a novel member of the Chlamydiales order.

Simkania negevensis is a Chlamydia-related bacterium discovered in 1993 and represents the founding member of the Simkaniaceae family within the Chlamydiales order. As other Chlamydiales, it is an obligate intracellular bacterium characterized by a biphasic developmental cycle. Its similarities with the pathogenic Chlamydia trachomatis and Chlamydia pneumoniae make it an interesting bacterium. So far, little is known about its biology, but S. negevensis harbors various microbiological characteristics of interest, including a strong association of the Simkania-containing vacuole with the ER and the presence of an intron in the 23S rRNA encoding gene. Evidence of human exposition has been reported worldwide. However, there is a lack of robust clinical studies evaluating its implication in human diseases; current data suggest an association with pneumonia and bronchiolitis making S. negevensis a potential emerging pathogen. Owing to its fastidious growth requirements, the clinical relevance of S. negevensis is probably underestimated. In this review, we summarize the current knowledge on S. negevensis and explore future research challenges.

Molecular Evidence of Chlamydia-Like Organisms in the Feces of Myotis daubentonii Bats.

Chlamydia-like organisms (CLOs) are recently identified members of the Chlamydiales order. CLOs share intracellular lifestyles and biphasic developmental cycles, and they have been detected in environmental samples as well as in various hosts such as amoebae and arthropods. In this study, we screened bat feces for the presence of CLOs by molecular analysis. Using pan-Chlamydiales PCR targeting the 16S rRNA gene, Chlamydiales DNA was detected in 54% of the specimens. PCR amplification, sequencing, and phylogenetic analysis of the 16S rRNA and 23S rRNA genes were used to classify positive specimens and infer their phylogenetic relationships. Most sequences matched best with Rhabdochlamydia species or uncultured Chlamydia sequences identified in ticks. Another set of sequences matched best with sequences of the Chlamydia genus or uncultured Chlamydiales from snakes. To gain evidence of whether CLOs in bat feces are merely diet borne, we analyzed insects trapped from the same location where the bats foraged. Interestingly, the CLO sequences resembling Rhabdochlamydia spp. were detected in insect material as well, but the other set of CLO sequences was not, suggesting that this set might not originate from prey. Thus, bats represent another potential host for Chlamydiales and could harbor novel, previously unidentified members of this order. IMPORTANCE: Several pathogenic viruses are known to colonize bats, and recent analyses indicate that bats are also reservoir hosts for bacterial genera. Chlamydia-like organisms (CLOs) have been detected in several animal species. CLOs have high 16S rRNA sequence similarity to Chlamydiaceae and exhibit similar intracellular lifestyles and biphasic developmental cycles. Our study describes the frequent occurrence of CLO DNA in bat feces, suggesting an expanding host species spectrum for the Chlamydiales As bats can acquire various infectious agents through their diet, prey insects were also studied. We identified CLO sequences in bats that matched best with sequences in prey insects but also CLO sequences not detected in prey insects. This suggests that a portion of CLO DNA present in bat feces is not prey borne. Furthermore, some sequences from bat droppings not originating from their diet might well represent novel, previously unidentified members of the Chlamydiales order.