ESCMID postgraduate technical workshop on intracellular bacteria: from biology to clinic.

Infection by intracellular bacteria can lead to several diseases in both veterinary and human medicine. Unfortunately, the biology of these intracellular bacteria is highly complex due to their interactions with their host cells. Thus, it is very important to develop several tools in order to better understand the complex intracellular life of these pathogens, so allowing to improve the diagnosis options and the treatments of infectious diseases that they are causing. The workshop organised in Villars-sur-Ollon (Switzerland) by the ESCMID Study group on intracellular bacteria was a good opportunity to enhance our knowledge on these fastidious pathogens. During 5 days, 15 speakers gave 41 talks, covering all fields, from biology to clinic of different intracellular bacteria such as Bartonella, Chlamydia, Coxiella, Ehrlichia, Listeria, Parachlamydia, Rickettsia, and Waddlia. The format of this postgraduate course, which took place in the Swiss mountains, allowed interactive sessions and living discussions between the participants coming from all around the world. One of the major strength was to gather epidemiologists, clinical microbiologists, infectious diseases specialists, entomologists, veterinarians as well as bioinformaticians, biochemists and biologists to deliver a unique "one-health science" on intracellular bacteria. Here, we summarise the main take-home messages delivered during this meeting

Mouse Model of Respiratory Tract Infection Induced by Waddlia chondrophila.

Waddlia chondrophila, an obligate intracellular bacterium belonging to the Chlamydiales order, is considered as an emerging pathogen. Some clinical studies highlighted a possible role of W. chondrophila in bronchiolitis, pneumonia and miscarriage. This pathogenic potential is further supported by the ability of W. chondrophila to infect and replicate within human pneumocytes, macrophages and endometrial cells. Considering that W. chondrophila might be a causative agent of respiratory tract infection, we developed a mouse model of respiratory tract infection to get insight into the pathogenesis of W. chondrophila. Following intranasal inoculation of 2 x 108 W. chondrophila, mice lost up to 40% of their body weight, and succumbed rapidly from infection with a death rate reaching 50% at day 4 post-inoculation. Bacterial loads, estimated by qPCR, increased from day 0 to day 3 post-infection and decreased thereafter in surviving mice. Bacterial growth was confirmed by detecting dividing bacteria using electron microscopy, and living bacteria were isolated from lungs 14 days post-infection. Immunohistochemistry and histopathology of infected lungs revealed the presence of bacteria associated with pneumonia characterized by an important multifocal inflammation. The high inflammatory score in the lungs was associated with the presence of pro-inflammatory cytokines in both serum and lungs at day 3 post-infection. This animal model supports the role of W. chondrophila as an agent of respiratory tract infection, and will help understanding the pathogenesis of this strict intracellular bacterium

Severe pneumonia due to Parachlamydia acanthamoebae following intranasal inoculation: a mice model.

Parachlamydia acanthamoebae is an obligate intracellular bacterium naturally infecting free-living amoebae. The role of this bacterium as an agent of pneumonia is suggested by sero-epidemiological studies and molecular surveys. Furthermore, P. acanthamoebae may escape macrophages microbicidal effectors. Recently, we demonstrated that intratracheal inoculation of P. acanthamoebae induced pneumonia in 100% of infected mice. However, the intratracheal route of infection is not the natural way of infection and we therefore developed an intranasal murine model. Mice inoculated with P. acanthamoebae by intranasal inoculation lost 18% of their weight up to 8 days post-inoculation. All mice presented histological signs of pneumonia at day 2, 4, 7, and 10 post-inoculation, whereas no control mice harboured signs of pneumonia. A 5-fold increase in bacterial load was observed from day 0 to day 4 post-inoculation. Lungs of inoculated mice were positive by Parachlamydia-specific immunohistochemistry 4 days post-inoculation, and P. acanthamoebae were localized within macrophages. Thus, we demonstrated that P. acanthamoebae induce a severe pneumonia in mice. This animal model (i) further supports the role of P. acanthamoebae as an agent of pneumonia, confirming the third Koch postulate, and (ii) identified alveolar macrophages as one of the initial cells where P. acanthamoebae is localized following infection

A predation assay using amoebae to screen for virulence factors unearthed the first W. chondrophila inclusion membrane protein.

Waddlia chondrophila is an intracellular bacterium phylogenetically related to the well-studied human and animal pathogens of the Chlamydiaceae family. In the last decade, W. chondrophila was convincingly demonstrated to be associated with adverse pregnancy outcomes in humans and abortions in animals. All members of the phylum Chlamydiae possess a Type Three Secretion System that they use for delivering virulence proteins into the host cell cytosol to modulate their environment and create optimal conditions to complete their life cycle. To identify W. chondrophila virulence proteins, we used an original screening approach that combines a cosmid library with an assay monitoring resistance to predation by phagocytic amoebae. This technique combined with bioinformatic data allowed the identification of 28 candidate virulence proteins, including Wimp1, the first identified inclusion membrane protein of W. chondrophila

Waddlia chondrophila induces systemic infection, organ pathology, and elicits Th1-associated humoral immunity in a murine model of genital infection.

Waddlia chondrophila is a known bovine abortigenic Chlamydia-related bacterium that has been associated with adverse pregnancy outcomes in human. However, there is a lack of knowledge regarding how W. chondrophila infection spreads, its ability to elicit an immune response and induce pathology. A murine model of genital infection was developed to investigate the pathogenicity and immune response associated with a W. chondrophila infection. Genital inoculation of the bacterial agent resulted in a dose-dependent infection that spread to lumbar lymph nodes and successively to spleen and liver. Bacterial-induced pathology peaked on day 14, characterized by leukocyte infiltration (uterine horn, liver, and spleen), necrosis (liver) and extramedullary hematopoiesis (spleen). Immunohistochemistry demonstrated the presence of a large number of W. chondrophila in the spleen on day 14. Robust IgG titers were detected by day 14 and remained high until day 52. IgG isotypes consisted of high IgG2a, moderate IgG3 and no detectable IgG1, indicating a Th1-associated immune response. This study provides the first evidence that W. chondrophila genital infection is capable of inducing a systemic infection that spreads to major organs, induces uterus, spleen, and liver pathology and elicits a Th1-skewed humoral response. This new animal model will help our understanding of the mechanisms related to intracellular bacteria-induced miscarriages, the most frequent complication of pregnancy that affects one in four women

Prevalence of <i>Anaplasma phagocytophilum</i> and <i>Coxiella burnetii</i> in <i>Ixodes ricinus</i> ticks in Switzerland: an underestimated epidemiologic risk.

Ticks are vectors of several microorganisms responsible for infectious diseases in human and animals, such as &lt;i&gt;Anaplasma phagocytophilum&lt;/i&gt; and &lt;i&gt;Coxiella burnetii.&lt;/i&gt; In this study, we investigated the prevalence of these two bacteria in 62 889 &lt;i&gt;Ixodes ricinus&lt;/i&gt; ticks in selected regions covering all Switzerland. A high prevalence of 11.9% of &lt;i&gt;A. phagocytophilum&lt;/i&gt; DNA was observed by real-time PCR on 8534 pools of ticks. This pool prevalence corresponds to an estimated prevalence of 1.71% in individual tick. A total of 144 of the 171 collection sites (84.2%) were positive for the presence of &lt;i&gt;A. phagocytophilum,&lt;/i&gt; and these sites were homogenously distributed throughout Switzerland. Such prevalence and geographical distribution underline the risk of human and animal exposure to &lt;i&gt;A. phagocytophilum&lt;/i&gt; and highlight the need to assess the epidemiology and clinical diagnosis of human and animal anaplasmosis in Switzerland. However, DNA of &lt;i&gt;C. burnetii&lt;/i&gt; was never found in any tick pool. This absence suggests a very low role of &lt;i&gt;I. ricinus&lt;/i&gt; ticks as vector and reservoir of &lt;i&gt;C. burnetii&lt;/i&gt; in Switzerland, and it supports previous reports demonstrating the role of sheep and goats in the epidemiology of Q fever. However, considering its pathogenic potential, it is necessary to keep monitoring for the possible reemergence of this bacterium in ticks in the future

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Scrub typhus ecology: a systematic review of Orientia in vectors and hosts

Abstract Scrub typhus, caused by Orientia tsutsugamushi, is an important and neglected vector-borne zoonotic disease with an expanding known distribution. The ecology of the disease is complex and poorly understood, impairing discussion of public health interventions. To highlight what we know and the themes of our ignorance, we conducted a systematic review of all studies investigating the pathogen in vectors and non-human hosts. A total of 276 articles in 7 languages were included, with 793 study sites across 30 countries. There was no time restriction for article inclusion, with the oldest published in 1924. Seventy-six potential vector species and 234 vertebrate host species were tested, accounting for over one million trombiculid mites (‘chiggers’) and 83,000 vertebrates. The proportion of O. tsutsugamushi positivity was recorded for different categories of laboratory test and host species. Vector and host collection sites were geocoded and mapped. Ecological data associated with these sites were summarised. A further 145 articles encompassing general themes of scrub typhus ecology were reviewed. These topics range from the life-cycle to transmission, habitats, seasonality and human risks. Important gaps in our understanding are highlighted together with possible tools to begin to unravel these. Many of the data reported are highly variable and inconsistent and minimum data reporting standards are proposed. With more recent reports of human Orientia sp. infection in the Middle East and South America and enormous advances in research technology over recent decades, this comprehensive review provides a detailed summary of work investigating this pathogen in vectors and non-human hosts and updates current understanding of the complex ecology of scrub typhus. A better understanding of scrub typhus ecology has important relevance to ongoing research into improving diagnostics, developing vaccines and identifying useful public health interventions to reduce the burden of the disease.</jats:p

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