Antibiotic susceptibility of Estrella lausannensis, a potential emerging pathogen.

Estrella lausannensis is a new Chlamydia-related bacterium, belonging to the Criblamydiaceae family. As suggested by its species name, this bacterium harbors a peculiar star shape. E. lausannensis is able to infect a wide range of amoebal, fish and mammalian cell lines. Moreover, seroprevalence of 2.9% was reported in children and in women with tubal pathology, showing that humans are commonly exposed to this recently discovered strict intracellular bacteria considered as a potential pathogen. Antibiotic susceptibility was determined using two approaches: qPCR and cellular mortality assay. Antibiotics classically used against intracellular bacteria were tested, including β-lactams, fluoroquinolones, cyclines and macrolides. We showed that E. lausannensis is resistant to β-lactams and fluoroquinolones, and sensitive to cyclines. Interestingly, E. lausannensis is slightly resistant to azithromycin with a MIC of 2 μg/ml, which is 10 fold higher compared to Waddlia chondrophila and Parachlamydia acanthamoebae MIC's. A single A2059C mutation in 23S rRNA gene could be responsible for this unexpected resistance.

Chlamydiaceae and Chlamydia-like organisms in free-living small mammals in Europe and Afghanistan.

Few data are available on the occurrence of chlamydial infections in wild small mammals. We investigated the significance of free-living small mammals as reservoirs or transmission hosts for microorganisms of the phylum/class Chlamydiae. We obtained 3,664 tissue samples from 911 animals in Switzerland, Germany, Austria, the Czech Republic, and Afghanistan. Samples included internal organs (n = 3,652) and feces (n = 12) from 679 rodents (order Rodentia) and 232 insectivores (order Soricomorpha) and were tested by three TaqMan® real-time PCRs specific for members of the family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia spp. and Waddlia spp. Only one of 911 (0.11%) animals exhibited a questionable positive result by Chlamydiaceae-specific real-time PCR. Five of 911 animals were positive by specific real-time PCR for Parachlamydia spp. but could not be confirmed by quantitative PCR targeting the Parachlamydia acanthamoebae secY gene (secY qPCR). One of 746 animals (0.13%) was positive by real-time PCR for Waddlia chondrophila. This result was confirmed by Waddlia secY qPCR. This is the first detection of Chlamydia-like organisms in small wildlife in Switzerland. Considering previous negative results for Chlamydiaceae in wild ruminant species from Switzerland, these data suggest that wild small mammals are unlikely to be important carriers or transport hosts for Chamydiaceae and Chlamydia-like organisms.

Parachlamydia acanthamoebae, an emerging agent of pneumonia.

Parachlamydia acanthamoebae is a Chlamydia-like organism that easily grows within Acanthamoeba spp. Thus, it probably uses these widespread free-living amoebae as a replicative niche, a cosmopolite aquatic reservoir and a vector. A potential role of P. acanthamoebae as an agent of lower respiratory tract infection was initially suggested by its isolation within an Acanthamoeba sp. recovered from the water of a humidifier during the investigation of an outbreak of fever. Additional serological and molecular-based investigations further supported its pathogenic role, mainly in bronchiolitis, bronchitis, aspiration pneumonia and community-acquired pneumonia. P. acanthamoebae was shown to survive and replicate within human macrophages, lung fibroblasts and pneumocytes. Moreover, this strict intracellular bacterium also causes severe pneumonia in experimentally infected mice, thus fulfilling the third and fourth Koch criteria for a pathogenic role. Consequently, new tools have been developed for the diagnosis of parachlamydial infections. It will be important to routinely search for this emerging agent of pneumonia, as P. acanthamoebae is apparently resistant to quinolones, which are antibiotics often used for the empirical treatment of atypical pneumonia. Other Chlamydia-related bacteria, including Protochlamydia naegleriophila, Simkania negevensis and Waddlia chondrophila, might also cause lung infections. Moreover, several additional novel chlamydiae, e.g. Criblamydia sequanensis and Rhabdochlamydia crassificans, have been discovered and are now being investigated for their human pathogenicity.

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage.

Chlamydiales and the innate immune response: friend or foe?

Pathogenicity of Chlamydia and Chlamydia-related bacteria could be partially mediated by an enhanced activation of the innate immune response. The study of this host pathogen interaction has proved challenging due to the restricted in vitro growth of these strict intracellular bacteria and the lack of genetic tools to manipulate their genomes. Despite these difficulties, the interactions of Chlamydiales with the innate immune cells and their effectors have been studied thoroughly. This review aims to point out the role of pattern recognition receptors and signal molecules (cytokines, reactive oxygen species) of the innate immune response in the pathogenesis of chlamydial infection. Besides inducing clearance of the bacteria, some of these effectors may be used by the Chlamydia to establish chronic infections or to spread. Thus, the induced innate immune response seems to be variable depending on the species and/or the serovar, making the pattern more complex. It remains crucial to determine the common players of the innate immune response in order to help define new treatment strategies and to develop effective vaccines. The excellent growth in phagocytic cells of some Chlamydia-related organisms such as Waddlia chondrophila supports their use as model organisms to study conserved features important for interactions between the innate immunity and Chlamydia.

Comparison of five commercial serological tests for the detection of anti-Chlamydia trachomatis antibodies.

Screening for Chlamydia trachomatis-specific antibodies is valuable in investigating recurrent miscarriage, tubal infertility and extrauterine pregnancy. We compared here the performance of immunofluorescence (IF) to four other commercial tests in detecting IgG antibodies directed against C. trachomatis: two enzyme-linked immunosorbent assays (ELISAs) using the major outer membrane protein (MOMP) as the antigen, commercialised respectively by Medac and R-Biopharm (RB), one ELISA using the chlamydial heat shock protein 60 (cHSP60) as the antigen (Medac), as well as a new automated epifluorescence immunoassay (InoDiag). A total of 405 patients with (n = 251) and without (n = 154) miscarriages were tested by all five tests. The prevalence of C. trachomatis-specific IgG antibodies as determined by the IF, cHSP60-Medac, MOMP-Medac, MOMP-RB and InoDiag was 14.3, 23.2, 14.3, 11.9 and 26.2%, respectively. InoDiag exhibited the highest sensitivity, whereas MOMP-RB showed the best specificity. Cross-reactivity was observed with C. pneumoniae using IF, MOMP-RB and InoDiag, and Parachlamydia acanthamoebae using the cHSP60 ELISA test. No cross-reactivity was observed between C. trachomatis and the other Chlamydiales (Neochlamydia hartmannellae, Waddlia chondrophila and Simkania negevensis). Given its high sensitivity, the new automated epifluorescence immunoassay from InoDiag represents an interesting alternative. The MOMP-based ELISA of R-Biopharm should be preferred for large serological studies, given the high throughput of ELISA and its excellent specificity.

Role of Chlamydia trachomatis and emerging Chlamydia-related bacteria in ectopic pregnancy in Vietnam.

In this case-control study, we investigated the seroprevalence and molecular evidence of Chlamydia trachomatis and Waddlia chondrophila in ectopic pregnancies (EP) and uneventful control pregnancies in 343 women from Vietnam. Whereas presence of C. trachomatis IgG was strongly associated with EP [adjusted odds ratio (aOR) 5·41, 95% confidence interval (CI) 2·58-11·32], its DNA remained undetected in all tubal lesions. We confirmed an independent association between antibodies against Waddlia and previous miscarriage (aOR 1·87, 95% CI 1·02-3·42). Further investigations are needed to understand the clinical significance of Waddlia's high seroprevalence (25·9% in control pregnancies) in this urban population.

Cell wall precursors are required to organize the chlamydial division septum.

Members of the Chlamydiales order are major bacterial pathogens that divide at mid-cell, without a sequence homologue of the FtsZ cytokinetic tubulin and without a classical peptidoglycan cell wall. Moreover, the spatiotemporal mechanisms directing constriction in Chlamydia are not known. Here we show that the MreB actin homologue and its conserved regulator RodZ localize to the division furrow in Waddlia chondrophila, a member of the Chlamydiales order implicated in human miscarriage. RodZ is recruited to the septal site earlier than MreB and in a manner that depends on biosynthesis of the peptidoglycan precursor lipid II by the MurA enzyme. By contrast, crosslinking of lipid II peptides by the Pbp3 transpeptidase disperses RodZ from the septum. Altogether, these findings provide a cytological framework for understanding chlamydial cytokinesis driven by septal cell wall synthesis.

The High Prevalence and Diversity of Chlamydiales DNA within Ixodes ricinus Ticks Suggest a Role for Ticks as Reservoirs and Vectors of Chlamydia-Related Bacteria.

The Chlamydiales order is composed of nine families of strictly intracellular bacteria. Among them, Chlamydia trachomatis, C. pneumoniae, and C. psittaci are established human pathogens, whereas Waddlia chondrophila and Parachlamydia acanthamoebae have emerged as new pathogens in humans. However, despite their medical importance, their biodiversity and ecology remain to be studied. Even if arthropods and, particularly, ticks are well known to be vectors of numerous infectious agents such as viruses and bacteria, virtually nothing is known about ticks and chlamydia. This study investigated the prevalence of Chlamydiae in ticks. Specifically, 62,889 Ixodes ricinus ticks, consolidated into 8,534 pools, were sampled in 172 collection sites throughout Switzerland and were investigated using pan-Chlamydiales quantitative PCR (qPCR) for the presence of Chlamydiales DNA. Among the pools, 543 (6.4%) gave positive results and the estimated prevalence in individual ticks was 0.89%. Among those pools with positive results, we obtained 16S rRNA sequences for 359 samples, allowing classification of Chlamydiales DNA at the family level. A high level of biodiversity was observed, since six of the nine families belonging to the Chlamydiales order were detected. Those most common were Parachlamydiaceae (33.1%) and Rhabdochlamydiaceae (29.2%). "Unclassified Chlamydiales" (31.8%) were also often detected. Thanks to the huge amount of Chlamydiales DNA recovered from ticks, this report opens up new perspectives on further work focusing on whole-genome sequencing to increase our knowledge about Chlamydiales biodiversity. This report of an epidemiological study also demonstrates the presence of Chlamydia-related bacteria within Ixodes ricinus ticks and suggests a role for ticks in the transmission of and as a reservoir for these emerging pathogenic Chlamydia-related bacteria.

Disassembly of a Medial Transenvelope Structure by Antibiotics during Intracellular Division.

Chlamydiales possess a minimal but functional peptidoglycan precursor biosynthetic and remodeling pathway involved in the assembly of the division septum by an atypical cytokinetic machine and cryptic or modified peptidoglycan-like structure (PGLS). How this reduced cytokinetic machine collectively coordinates the invagination of the envelope has not yet been explored in Chlamydiales. In other Gram-negative bacteria, peptidoglycan provides anchor points that connect the outer membrane to the peptidoglycan during constriction using the Pal-Tol complex. Purifying PGLS and associated proteins from the chlamydial pathogen Waddlia chondrophila, we unearthed the Pal protein as a peptidoglycan-binding protein that localizes to the chlamydial division septum along with other components of the Pal-Tol complex. Together, our PGLS characterization and peptidoglycan-binding assays support the notion that diaminopimelic acid is an important determinant recruiting Pal to the division plane to coordinate the invagination of all envelope layers with the conserved Pal-Tol complex, even during osmotically protected intracellular growth.

Trafficking of Estrella lausannensis in human macrophages.

Estrella lausannensis is a new member of the Chlamydiales order. Like other Chlamydia-related bacteria, it is able to replicate in amoebae and in fish cell lines. A preliminary study investigating the pathogenic potential of Chlamydia-related bacteria found a correlation between antibody response to E. lausannensis and pneumonia in children. To further investigate the pathogenic potential of E. lausannensis, we determined its ability to grow in human macrophages and its intracellular trafficking. The replication in macrophages resulted in viable E. lausannensis; however, it caused a significant cytopathic effect. The intracellular trafficking of E. lausannensis was analyzed by determining the interaction of the Estrella-containing inclusions with various endocytic markers as well as host organelles. The E. lausannensis inclusion escaped the endocytic pathway rapidly avoiding maturation into phagolysosomes by preventing both EEA-1 and LAMP-1 accumulation. Compared to Waddlia chondrophila, another Chlamydia-related bacteria, the recruitment of mitochondria and endoplasmic reticulum was minimal for E. lausannensis inclusions. Estrella lausannensis appears to use a distinct source of nutrients and energy compared to other members of the Chlamydiales order. In conclusion, we hypothesize that E. lausannensis has a restricted growth in human macrophages, due to its reduced capacity to control programmed cell death.

Importance of amoebae as a tool to isolate amoeba-resisting microorganisms and for their ecology and evolution: the Chlamydia paradigm.

Free-living amoebae are distributed worldwide and are frequently in contact with humans and animals. As cysts, they can survive in very harsh conditions and resist biocides and most disinfection procedures. Several microorganisms, called amoeba-resisting microorganisms (ARMs), have evolved to survive and multiply within these protozoa. Among them are many important pathogens, such as Legionella and Mycobacteria, and also several newly discovered Chlamydia-related bacteria, such as Parachlamydia acanthamoebae, Estrella lausannensis, Simkania negevensis or Waddlia chondrophila whose pathogenic role towards human or animal is strongly suspected. Amoebae represent an evolutionary crib for their resistant microorganisms since they can exchange genetic material with other ARMs and develop virulence traits that will be further used to infect other professional phagocytes. Moreover, amoebae constitute an ideal tool to isolate strict intracellular microorganisms from complex microbiota, since they will feed on other fast-growing bacteria, such as coliforms potentially present in the investigated samples. The paradigm that ARMs are likely resistant to macrophages, another phagocytic cell, and that they are likely virulent towards humans and animals is only partially true. Indeed, we provide examples of the Chlamydiales order that challenge this assumption and suggest that the ability to multiply in protozoa does not strictly correlate with pathogenicity and that we should rather use the ability to replicate in multiple and diverse eukaryotic cells as an indirect marker of virulence towards mammals. Thus, cell-culture-based microbial culturomics should be used in the future to try to discover new pathogenic bacterial species.

Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion.

Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion.

Waddlia, Parachlamydia and Chlamydiaceae in bovine abortion.

The etiology remains unknown in many cases of bovine abortion in Switzerland. Bacteria of the Chlamydiales order are known abortive agents, therefore cases of bovine abortion from three representative regions of Switzerland were investigated in this study. Particularly Chlamydiaceae as well as the Chlamydia-like organisms Waddlia and Parachlamydia were of interest, especially because of their possible zoonotic potential. Placenta samples (n=343) were tested for these bacteria by different PCR-methods, immunohistochemistry and serology for Chlamydia abortus. Additionally an attempt for the isolation of Waddlia and Parachlamydia was made by co-cultivation in amoebae. In 67.3% of the 343 cases a necrotizing and/or purulent placentitis was found histologically. By real-time PCR, 0.9% (3/343) of the cases were positive for Waddlia, 13.4% (46/343) positive for Parachlamydia and 14.6% (50/343) positive or questionable positive for Chlamydiaceae. Of these samples, confirmation by immunohistochemistry was possible in 2/3 cases for Waddlia, 25/46 for Parachlamydia and 4/50 for Chlamydiaceae. Of the 50 cases positive or questionable positive for Chlamydiaceae, species-identification by ArrayTube Microarray or 16S rRNA PCR resulted in 41 cases positive for C. abortus whereas the presence of Chlamydia suis was confirmed in four and Chlamydia pecorum in one case. This study brought evidence for the importance of different members of Chlamydiales in different regions of Switzerland although Waddlia is not occurring in a high prevalence. On the other hand mixed infections with different Chlamydiales as well as with other abortigenic agents could be found.