Sébastien Aeby

Development of a New Chlamydiales-Specific Real-Time PCR and Its Application to Respiratory Clinical Samples

ABSTRACT Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family.

Parachlamydia and Rhabdochlamydia in Premature Neonates

To the Editor: New members have recently been recognized in the order Chlamydiales (1). The family Rhabdochlamydiaceae includes R. porcellionis (a parasite of Porcellio scaber) and R. crassificans (a pathogen of the cockroach Blatta orientalis) (2,3); their pathogenic role in humans has not yet been investigated. Parachlamydia acanthamoebae and Protochlamydia naegleriophila belong to the family Parachlamydiaceae (1,4). Increasing evidence indicates that these obligate intracellular bacteria infecting free-living amebae may cause respiratory diseases in humans (1). Recent findings also suggest a role for Parachlamydia in miscarriage, stillbirth, and preterm labor (5–7). Whether these bacteria may contaminate the newborns of infected mothers is unknown. The aims of this study were to 1) develop a real-time PCR for detecting Rhabdochlamydia spp. and 2) apply this PCR, and those previously described for Parachlamydia and Protochlamydia (4,8), to respiratory samples from premature neonates. Using the GenBank database (www.ncbi.nlm.nih.gov), we selected primers RcF (5′-GACGCTGCGTGAGTGATGA-3′) and RcR (5′-CCGGTGCTTCTTTACGCAGTA-3′), and probe RcS (5′-6 carboxyfluorescein-CTTTCGGGTTGTAAAACTCTTTCGCGCA-Black Hole Quencher 1-3′), which amplify parts of the 16S rRNA encoding gene, to specifically amplify Rhabdochlamydia spp. The 5′-FAM probe (Eurogentec, Seraing, Belgium) contained locked nucleic acids (underlined) to improve specificity. Reactions were performed with 0.2 μM of each primer, 0.1 μM of probe, and iTaq Supermix (Bio-Rad, Rheinach, Switzerland). PCR products were detected with ABI Prism 7000 (Applied Biosystems, Rotkreuz, Switzerland). Inhibition, negative PCR mixture, and extraction controls were systematically tested. To enable quantification, a plasmid containing the target gene was constructed as described (4,9). The analytical sensitivity of the real-time PCR for Rhabdochlamydia spp. was <10 copies DNA/μL. No cross-amplification was observed when the analytical specificity was tested with human, amebal (Acanthamoeba castellanii ATCC 30010), and bacterial DNA (Technical Appendix). Intrarun and interrun reproducibility were excellent (Technical Appendix). This PCR and those previously described for Parachlamydia and Protochlamydia (4,8) were retrospectively applied to 39 respiratory samples from 29 neonates admitted in the neonatology unit of our institution (median 1 sample per patient, range 1–4 sample). All but 1 patient had a gestational age at birth <36 weeks (median 28.6, range 24.6–41.2 weeks). Respiratory distress syndrome was present in 25 (86%) of these 29 neonates. Samples had been drawn a median of 14 days (range 1–229 days) after birth, when clinically indicated. Results of PCR for Parachlamydia, Protochlamydia, and Rhabdochlamydia were positive for 9 (31%), 0 (0%), and 4 (14%) neonates, respectively. Positive results were obtained on the first sample drawn after birth for all but 2 neonates (initial negative results). One patient had positive PCR results for Parachlamydia and Rhabdochlamydia. These 12 newborns with positive PCR results for Parachlamydia and/or Rhabdochlamydia were compared with the 17 who had negative PCR results (Table). Table Characteristics of 29 newborns with positive PCR results for Parachlamydia acanthamoebae or Rhabdochlamydia spp. and controls* Newborns with a Chlamydia-related organism documented in the respiratory tract had a significantly worse primary adaptation score (Apgar). These patients experienced more resuscitation maneuvers at birth. Durations of invasive mechanical ventilation and hospital stay were also longer among them. Three newborns died, compared with no deaths among the 17 with negative PCR results (p = 0.06). Pneumonia was documented in 5 of the 12 patients with positive Parachlamydia and/or Rhabdochlamydia PCR results but was concomitant to PCR positivity for only 3 of them. An alternative etiology was documented in all 3 (Technical Appendix). Parachlamydia and Rhabdochlamydia have thus been detected in a population of premature neonates. Most of these patients had severe respiratory distress syndrome, and the role of these bacteria as a causal agent of pneumonia could not be clearly assessed. The longer duration of mechanical ventilation for newborns with positive PCR results may suggest an occult superinfection with a Chlamydia-related bacterium contributing to the severity of the initial respiratory disease. Our results also raise a question about the mode of acquisition of these microorganisms. A recent study reported a higher seroprevalence of Parachlamydia in women experiencing miscarriage (5,6), and DNA of this bacterium has been detected in the amniotic fluid of a woman with premature delivery (7). Whether neonatal infection results from a systemic infection during pregnancy or an inoculation at delivery is unknown. Because of the retrospective design of the study, no samples from the mothers were available for additional molecular or serologic analyses. Hospital water supplies are an important reservoir of free-living amebae and may represent another mode of acquisition because patients undergoing mechanical ventilation are exposed to aerosolized particles (10). Simultaneous detection of Parachlamydia and Rhabdochlamydia in 2 patients with initial negative results and their simultaneous detection in 1 neonate supports the latter hypothesis. In conclusion, Parachlamydia and Rhabdochlamydia DNA were detected in respiratory secretions of premature newborns with more severe conditions at birth, more mechanical ventilation requirements, and a trend toward a higher mortality rate. The pathogenic role of these Chlamydia-related bacteria in neonates deserves further investigations.

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.

ABSTRACT 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.

Presence of Chlamydiales DNA in ticks and fleas suggests that ticks are carriers of Chlamydiae.

The Chlamydiales order includes the Chlamydiaceae, Parachlamydiaceae, Waddliaceae, Simkaniaceae, Criblamydiaceae, Rhabdochlamydiaceae, Clavichlamydiaceae, and Piscichlamydiaceae families. Members of the Chlamydiales order are obligate intracellular bacteria that replicate within eukaryotic cells of different origins including humans, animals, and amoebae. Many of these bacteria are pathogens or emerging pathogens of both humans and animals, but their true diversity is largely underestimated, and their ecology remains to be investigated. Considering their potential threat on human health, it is important to expand our knowledge on the diversity of Chlamydiae, but also to define the host range colonized by these bacteria. Thus, using a new pan-Chlamydiales PCR, we analyzed the prevalence of Chlamydiales DNA in ticks and fleas, which are important vectors of several viral and bacterial infectious diseases. To conduct this study, 1340 Ixodes ricinus ticks prepared in 192 pools were collected in Switzerland and 55 other ticks belonging to different tick species and 97 fleas belonging to different flea species were harvested in Algeria. In Switzerland, the prevalence of Chlamydiales DNA in the 192 pools was equal to 28.1% (54/192) which represents an estimated prevalence in the 1340 individual ticks of between 4.0% and 28.4%. The pan-Chlamydiales qPCR was positive for 45% (25/55) of tick samples collected in Algeria. The sequencing of the positive qPCR amplicons revealed a high diversity of Chlamydiales species. Most of them belonged to the Rhabdochlamydiaceae and Parachlamydiaceae families. Thus, ticks may carry Chlamydiales and should thus be considered as possible vectors for Chlamydiales propagation to both humans and animals.

Discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

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 Criblamydiasequanensis, Estrellalausannensis, Parachlamydiaacanthamoebae, and Waddliachondrophila. 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.

Parachlamydia acanthamoebae in domestic cats with and without corneal disease

Corneal samples of cats with and without corneal diseases were screened with a pan-Chlamydiales PCR and specific PCRs for Parachlamydia, Protochlamydia, Chlamydophila felis, Acanthamoeba and feline herpesviruses (FHV-1). Several corneal samples tested positive for Parachlamydia and related Chlamydiales, indicating cat exposure to these intracellular bacteria.

Deer as a potential wildlife reservoir for Parachlamydia species

Wildlife populations represent an important reservoir for emerging pathogens and trans-boundary livestock diseases. However, detailed information relating to the occurrence of endemic pathogens such as those of the order Chlamydiales in such populations is lacking. During the hunting season of 2008, 863 samples (including blood, conjunctival swabs, internal organs and faeces) were collected in the Eastern Swiss Alps from 99 free-living red deer (Cervus elaphus) and 64 free-living roe deer (Capreolus capreolus) and tested using ELISA, PCR and immunohistochemistry for members of the family Chlamydiaceae and the genus Parachlamydia. Parachlamydia spp. were detected in the conjunctival swabs, faeces and internal organs of both species of deer (2.4% positive, with a further 29.5% inconclusive). The very low occurrence of Chlamydiaceae (2.5%) was in line with serological data (0.7% seroprevalence for Chlamydia abortus). Further investigations are required to elucidate the zoonotic potential, pathogenicity, and distribution of Parachlamydia spp. in wild ruminants.

β-Klotho deficiency protects against obesity through a crosstalk between liver, microbiota, and brown adipose tissue

β-Klotho (encoded by Klb) is the obligate coreceptor mediating FGF21 and FGF15/19 signaling. Klb-/- mice are refractory to beneficial action of pharmacological FGF21 treatment including stimulation of glucose utilization and thermogenesis. Here, we investigated the energy homeostasis in Klb-/- mice on high-fat diet in order to better understand the consequences of abrogating both endogenous FGF15/19 and FGF21 signaling during caloric overload. Surprisingly, Klb-/- mice are resistant to diet-induced obesity (DIO) owing to enhanced energy expenditure and BAT activity. Klb-/- mice exhibited not only an increase but also a shift in bile acid (BA) composition featured by activation of the classical (neutral) BA synthesis pathway at the expense of the alternative (acidic) pathway. High hepatic production of cholic acid (CA) results in a large excess of microbiota-derived deoxycholic acid (DCA). DCA is specifically responsible for activating the TGR5 receptor that stimulates BAT thermogenic activity. In fact, combined gene deletion of Klb and Tgr5 or antibiotic treatment abrogating bacterial conversion of CA into DCA both abolish DIO resistance in Klb-/- mice. These results suggested that DIO resistance in Klb-/- mice is caused by high levels of DCA, signaling through the TGR5 receptor. These data also demonstrated that gut microbiota can regulate host thermogenesis via conversion of primary into secondary BA. Pharmacologic or nutritional approaches to selectively modulate BA composition may be a promising target for treating metabolic disorders.

Neglected zoonotic agents in cattle abortion: molecular and serological screening of difficult to grow bacteria

Coxiella burnetii, Chlamydia abortus and Leptospira spp., are three agents that may lead to bovine abortion. The importance of these difficult to grow zoonotic bacterial pathogens lies in significant economic loss in animal production and the public health risk, at least in endemic countries. Routine bacteriological diagnostics of abortion in cattle in Switzerland is regulated by law including screening by serology and staining. However, only few infectious agents are examined using molecular approaches due to the high costs associated with extended analyses. In the present work, we used both serological and molecular methods to assess the possible role of these pathogens in bovine abortion. From 249 studied bovine abortion events, 242 placenta samples, 57 fetal abomasal content and 182 sera were taken from mother cows. The seroprevalence was 15.93%, 38.46% and 21.43% for C. burnetii (ELISA), C. abortus (ELISA) and pathogenic Leptospira spp. (microscopic agglutination test), respectively. Using specific real time PCR, the prevalence of C. burnetii, Chlamydiales and pathogenic Leptospira spp., were of 12.15%, 16.87% and 8.24%, respectively. After direct sequencing of Chlamydiales positive samples, we identified C. abortus in 8.84% of the cases and probable infection with Chlamydia-related bacteria in 5.22% of the cases. Altogether, routine abortion diagnostics did not detect a possible bacterial agent in 96 cases. Extending the spectrum of analysis could assign at least one possible abortive agent in 39 more cases. In conclusion, diagnostic approaches enabling the detection of C. burnetii, C. abortus and Leptospira spp., should be used more commonly due to their zoonotic potential.