Didier Raoult

Rickettsial evolution in the light of comparative genomics

Rickettsia are best known as strictly intracellular vector‐borne bacteria that cause mild to severe diseases in humans and other animals. Recent advances in molecular tools and biological experiments have unveiled a wide diversity of Rickettsia spp. that include species with a broad host range and some species that act as endosymbiotic associates. Molecular phylogenies of Rickettsia spp. contain some ambiguities, such as the position of R. canadensis and relationships within the spotted fever group. In the modern era of genomics, with an ever‐increasing number of sequenced genomes, there is enhanced interest in the use of whole‐genome sequences to understand pathogenesis and assess evolutionary relationships among rickettsial species. Rickettsia have small genomes (1.1–1.5 Mb) as a result of reductive evolution. These genomes contain split genes, gene remnants and pseudogenes that, owing to the colinearity of some rickettsial genomes, may represent different steps of the genome degradation process. Genomics reveal extreme genome reduction and massive gene loss in highly vertebrate‐pathogenic Rickettsia compared to less virulent or endosymbiotic species. Information gleaned from rickettsial genomics challenges traditional concepts of pathogenesis that focused primarily on the acquisition of virulence factors. Another intriguing phenomenon about the reduced rickettsial genomes concerns the large fraction of non‐coding DNA and possible functionality of these “non‐coding” sequences, because of the high conservation of these regions. Despite genome streamlining, Rickettsia spp. contain gene families, selfish DNA, repeat palindromic elements and genes encoding eukaryotic‐like motifs. These features participate in sequence and functional diversity and may play a crucial role in adaptation to the host cell and pathogenesis. Genome analyses have identified a large fraction of mobile genetic elements, including plasmids, suggesting the possibility of lateral gene transfer in these intracellular bacteria. Phylogenetic analyses have identified several candidates for horizontal gene acquisition among Rickettsia spp. including tra, pat2, and genes encoding for the type IV secretion system and ATP/ADP translocase that may have been acquired from bacteria living in amoebae. Gene loss, gene duplication, DNA repeats and lateral gene transfer all have shaped rickettsial genome evolution. A comprehensive analysis of the entire genome, including genes and non‐coding DNA, will help to unlock the mysteries of rickettsial evolution and pathogenesis.

Massive comparative genomic analysis reveals convergent evolution of specialized bacteria

BackgroundGenome size and gene content in bacteria are associated with their lifestyles. Obligate intracellular bacteria (i.e., mutualists and parasites) have small genomes that derived from larger free-living bacterial ancestors; however, the different steps of bacterial specialization from free-living to intracellular lifestyle have not been studied comprehensively. The growing number of available sequenced genomes makes it possible to perform a statistical comparative analysis of 317 genomes from bacteria with different lifestyles.ResultsCompared to free-living bacteria, host-dependent bacteria exhibit fewer rRNA genes, more split rRNA operons and fewer transcriptional regulators, linked to slower growth rates. We found a function-dependent and non-random loss of the same 100 orthologous genes in all obligate intracellular bacteria. Thus, we showed that obligate intracellular bacteria from different phyla are converging according to their lifestyle. Their specialization is an irreversible phenomenon characterized by translation modification and massive gene loss, including the loss of transcriptional regulators. Although both mutualists and parasites converge by genome reduction, these obligate intracellular bacteria have lost distinct sets of genes in the context of their specific host associations: mutualists have significantly more genes that enable nutrient provisioning whereas parasites have genes that encode Types II, IV, and VI secretion pathways.ConclusionOur findings suggest that gene loss, rather than acquisition of virulence factors, has been a driving force in the adaptation of parasites to eukaryotic cells. This comparative genomic analysis helps to explore the strategies by which obligate intracellular genomes specialize to particular host-associations and contributes to advance our knowledge about the mechanisms of bacterial evolution.ReviewersThis article was reviewed by Eugene V. Koonin, Nicolas Galtier, and Jeremy Selengut.

Bacterial genome sequencing and its use in infectious diseases.

The availability of genome sequences is revolutionising the fields of bacteriology and infectious diseases. By mid-2007, 479 bacterial genomes from 352 distinct species have been sequenced, including representatives of all notable human pathogens. Additionally, the genomes of several strains from each of 55 species have been sequenced. This tremendous amount of genomic data has led to unprecedented advances in pathogen diagnosis, genotyping, detection of virulence, and detection of resistance to antibiotics. We review current achievements in these fields and potential developments in the future for the clinical microbiology laboratory.

Adipose Tissue Serves as a Reservoir for Recrudescent Rickettsia prowazekii Infection in a Mouse Model

Brill-Zinsser disease, the relapsing form of epidemic typhus, typically occurs in a susceptible host years or decades after the primary infection; however, the mechanisms of reactivation and the cellular reservoir during latency are poorly understood. Herein we describe a murine model for Brill-Zinsser disease, and use PCR and cell culture to show transient rickettsemia in mice treated with dexamethasone >3 months after clinical recovery from the primary infection. Treatment of similarly infected mice with cyclosporine failed to produce recrudescent bacteremia. Therapy with doxycycline for the primary infection prevented recrudescent bacteremia in most of these mice following treatment with dexamethasone. Rickettsia prowazekii (the etiologic agent of epidemic typhus) was detected by PCR, cell culture, and immunostaining methods in murine adipose tissue, but not in liver, spleen, lung, or central nervous system tissues of mice 4 months after recovery from the primary infection. The lungs of dexamethasone-treated mice showed impaired expression of β-defensin transcripts that may be involved in the pathogenesis of pulmonary lesions. In vitro, R. prowazekii rickettsiae infected and replicated in the murine adipocyte cell line 3T3-L1. Collectively these data suggest a role for adipose tissue as a potential reservoir for dormant infections with R. prowazekii.

Coxiella burnetii infection of pseudoaneurysm of an aortic bypass graft with contiguous vertebral osteomyelitis

We report the case of a 67-year-old man who had an infection of a pseudoaneurysm of an aortic graft with contiguous vertebral osteomyelitis. The infectious organism was identified as Coxiella burnetii, a strict intracellular pathogen causing Q fever infection in humans. The patient was treated successfully with removal of the infected material in conjunction with extraanatomic bypass and specific antibiotic therapy. He is doing well after more than 3 years, with no evidence of recurrent periaortic infection on successive computed tomographic scans. We suspect that C. burnetii vascular graft infections could be underdiagnosed, because this cause is not frequently evoked. We suggest that extending the etiologic search to C. burnetii could decrease the number of undocumented vascular graft infections.

Relevance of the positron emission tomography in the diagnosis of vascular graft infection with Coxiella burnetii.

Coxiella burnetii, the causative agent of Q fever, may cause culture-negative vascular graft infections that can be diagnosed by serology and molecular biology. We present a case of vascular graft infection detected by positron emission tomography (PET) scanner. The presence of C. burnetii was confirmed by high antibody titers and positive polymerase chain reaction specific for C. burnetii. This report emphasizes the relevance of the PET scanner in the diagnosis of infection when used in association with Q fever serology and molecular biology for the etiological identification of C. burnetii.

Airways colonizations in patients undergoing lung cancer surgery

Lung cancer remains the main leading cancer-related cause of death in the world. For early-stage tumor, surgery stands out as the best curative option offering the greatest chance for cure. Despite improvement of per- and postoperative management, surgery continues to carry a high morbidity with a significant mortality. Among postoperative complications, respiratory failures (nosocomial pneumonia and acute respiratory distress syndrome) are currently the most frequent and serious, as well as being the primary cause of hospital death, after a lung resection for cancer. Because infectious etiologies have been highly incriminated in the development of these pulmonary complications, microbial airways colonizations (AWCs) are supposed to be an essential first step in the pathogenesis of these failures occurring in hospitalized and chronically ill individuals. These patients fulfill all the predisposing factors to bronchial colonizations and are particularly exposed to the development of respiratory failures in the postoperative setting, when secretion clearance and cough reflex are impaired. Under immunosuppressive conditions, AWC should act in a manner that increases its ability to stimulate microorganisms and increase the risks of superimposed infections. Few studies have addressed the problem of AWCs in patients submitted for lung cancer surgery. Because of several limitations, especially the lack of exhaustive microbiological studies, the conclusions that can be reached remain inconclusive. This review aims to report the existing literature on this critical and controversial issue, focusing on their specific incidence, their predisposing factors, their correlation with development of respiratory failures, and, in turn, the reliability of the current antibiotic prophylaxis for their prevention.

Q fever osteomyelitis: A case report and literature review

Q fever is a worldwide zoonosis caused by Coxiella burnetii. The clinical manifestations of Q fever include endocarditis, pneumonitis and hepatitis. Disease awareness and evolving diagnostic tests have enabled the recognition of unusual manifestations of Q fever. We report a case of Q fever osteomyelitis. A 51-year-old patient was admitted to hospital because of fever, leg weakness, and asthenia. His past medical history included surgery and a bone graft for the treatment of a giant cell tumor on the distal part of the femur. Blood and bone biopsy cultures were negative. Bone histological examination was consistent with a sub-acute or chronic inflammatory reaction that involved foci of epithelioid and gigantocellular infiltrates and necrosis. Serology testing revealed high antibody titers to C. burnetii antigens (phase I: IgG 3200; IgA 200; phase II: IgG 6400; IgA 400), which is indicative of chronic Q fever. The specific Polymerase Chain Reaction (PCR) of the abscess sample from the femoral region was positive for C. burnetii. The patient was treated for chronic Q fever with doxycycline and hydroxychloroquine for 18 months and recovered gradually without recurrence of pain or functional impairment. Q fever osteomyelitis is a rare and most likely underestimated disease. Epithelioid and gigantocellular granulomatous osteomyelitis in the context of culture-negative bone specimens should raise suspicion of Q fever. Serological tests, specific PCR and cell culture can provide evidence of a C. burnetii infection. Although bone diffusion may be a concern, the currently recommended treatment for Q fever was effective in this case.

The first reported cases of Q fever endocarditis in Thailand

We describe the first two reported cases of Q fever endocarditis in Thailand. Both patients were male, had pre-existing heart valve damage and had contact with cattle. Heightened awareness of Q fever could improve diagnosis and case management and stimulate efforts to identify risk factors and preventive measures.

Molecular Detection of Past Pathogens

Detection and characterisation of DNA is the most widely used approach for the study of past pathogens. This approach can be applied to various specimens, including environmental, vector and animal reservoir specimens as well as human corpses. Experimental data indicated that host-associated microbial DNA can survive for 20,000 years, and bacterial DNA preserved in permafrost specimens has been dated up to 1 million years. Current protocols targeted one pathogen at a time and universal 16S rDNA-based detection of bacteria have yielded ambiguous results. There is no universal detection of ancient virus so far. Major human patho- gens, e.g. Mycobacterium tuberculosis, Mycobacterium leprae, Yersinia pestis, Rickettsia prowazekii, Bartonella spp. and Spanish influenza virus have been detected in suitable human specimens. Ancient M. tuberculosis and Y. pestis organ- isms have been genotyped, whereas the entire RNA genome of Spanish influenza virus was reconstituted for extensive studies. Metagenomic approaches based on high throughput pyrosequencing may help further resolve forthcoming issues. Interpretation of experimental data has to be based upon strict rules due to potential contamination of specimens.