Alterations in gut microbiota composition under antibiotic pressure have been widely studied, revealing a restricted diversity of gut flora, including colonization by organisms such as Enterococci, while their impact on bacterial load is variable. High-level colonization by Akkermansia muciniphila, ranging from 39% to 84% of the total bacterial population, has been recently reported in two patients being treated with broad-spectrum antibiotics, although attempts to cultivate this microorganism have been unsuccessful.
Here, we propose an original approach of genome sequencing for Akkermansia muciniphila directly from the stool sample collected from one of these patients. We performed and assembly using metagenomic data obtained from the stool sample. We used a mapping method consisting of aligning metagenomic sequencing reads against the reference genome of the Akkermansia muciniphila MucT strain, and a De novo assembly to support this mapping method. We obtained draft genome of the Akkermansia muciniphila strain Urmite with only 56 gaps. The absence of particular metabolic requirement as possible explanation of our inability to culture this microorganism, suggests that the bacterium was dead before the inoculation of the stool sample. Additional antibiotic resistance genes were found following comparison with the reference genome, providing some clues pertaining to its survival and colonization in the gut of a patient treated with broad-spectrum antimicrobial agents. However, no gene coding for imipenem resistance was detected, although this antibiotic was a part of the patient’s antibiotic regimen.
This work highlights the potential of metagenomics to facilitate the assembly of genomes directly from human stool.
This article was reviewed by Eric Bapteste, William Martin and Vivek Anantharaman.
Electronic supplementary material
The online version of this article (doi:10.1186/s13062-015-0041-1) contains supplementary material, which is available to authorized users.
Akkermansia muciniphila; Genome; Gut microbiota; Metagenomics; Antibiotics
Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases.
The objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels.
Taken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns.
Tick-borne rickettsioses include mild to life-threatening diseases in humans worldwide. When removing an attached tick from the human body, patients and physicians may have two questions: 1) is the tick a known vector of a human infectious disease, and 2) is the tick infected by a pathogenic agent that could have been transmitted during the attachment period? The morphological identification of Ticks is difficult, and requires expertise and specific documentation. The use of Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged as an effective, rapid and inexpensive tool to identify arthropods including Ticks. Here, we show the utility of MALDI-TOF MS for the dual identification of tick species and the rapid detection of Rickettsia spp in Ticks. Such results can be used to guide decisions related to specific patient monitoring or the administration of preventive treatment. Additionally, the low consumable costs, the minimum time required for sample preparation and the rapid availability of the results of MALDI-TOF MS could be useful for epidemiological studies and tick-borne disease monitoring via the dual identification of vectors and the pathogens they carry in one step. These results present new opportunities for the management of other vector-borne diseases that are of importance to public health.
Murine typhus case was initially identified in Reunion, France, in 2012 in a tourist. Our investigation confirmed 8 autochthonous cases that occurred during January 2011–January 2013 in Reunion. Murine typhus should be considered in local patients and in travelers returning from Reunion who have fevers of unknown origin.
Fleas; murine typhus; Reunion; rodents; bacteria; zoonoses; vector-borne infections; France
Orientia tsutsugamushi; bronchoalveolar lavage; acute respiratory distress syndrome; France; Laos; lung; scrub typhus
Oropharyngeal swabs collected from patients with Q fever from France and from febrile patients from Senegal were tested by molecular assays for Coxiella burnetii. One positive result (0.08%) occurred for only one patient with acute Q fever. Throat swabs cannot replace blood serum samples as diagnostic tools for Q fever.
Ticks, belonging to the soft ticks species Ornithodorus sonrai, have been collected from six sites in Senegal and were tested for the presence of Bartonella spp. Initial screening by PCR revealed the presence of these bacteria in ticks from two villages, Soulkhou Thissé (5/8, 62.5%) and Maka Gouye (1/24, 4.2%). Three bacterial strains were isolated from live ticks, and the genetic characterization of these strains suggests that they belong to two previously unknown species. The pathogenicity of these two new species of Bartonella is not yet known. The new isolates described here are the first strains of Bartonella spp. from soft ticks and the first isolates from any arthropod species in Africa.
Ornithodorus sonrai; Soft ticks; Bartonella; Senegal
Bacillus simplex strain P558 was isolated from a fecal sample of a 25-year-old Saudi male. We sequenced the 5.98-Mb genome of the strain and compared it to that of B. simplex strain 1NLA3E.
The identification of mosquito vectors is generally based on morphological criteria, but for aquatic stages, morphological characteristics may be missing, leading to incomplete or incorrect identification. The high cost of molecular biology techniques requires the development of an alternative strategy. In the last decade, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has proved to be efficient for arthropod identification at the species level.
To investigate the usefulness of MALDI-TOF MS for the identification of mosquitoes at aquatic stages, optimizations of sample preparation, diet, body parts and storage conditions were tested. Protein extracts of whole specimens from second larval stage to pupae were selected for the creation of a reference spectra database. The database included a total of 95 laboratory-reared specimens of 6 mosquito species, including Anopheles gambiae (S form), Anopheles coluzzi (M form), Culex pipiens pipiens, Culex pipiens molestus, Aedes aegypti and 2 colonies of Aedes albopictus.
The present study revealed that whole specimens at aquatic stages produced reproducible and singular spectra according to the mosquito species. Moreover, MS protein profiles appeared weakly affected by the diet provided. Despite the low diversity of some MS profiles, notably for cryptic species, clustering analyses correctly classified all specimens tested at the species level followed by the clustering of early vs. late aquatic developmental stages. Discriminant mass peaks were recorded for the 6 mosquito species analyzed at larval stage 3 and the pupal stage. Querying against the reference spectra database of 149 new specimens at different aquatic stages from the 6 mosquito species revealed that 147 specimens were correctly identified at the species level and that early and late developmental stages were also distinguished.
The present work highlights that MALDI-TOF MS profiling may be useful for the rapid and reliable identification of mosquito species at aquatic stages. With this proteomic tool, it becomes now conceivable to survey mosquito breeding sites prior to the mosquitoes’ emergence and to adapt anti-vectorial measures according to the mosquito fauna detected.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-014-0544-0) contains supplementary material, which is available to authorized users.
MALDI-TOF mass spectrometry; Culicidae; Aquatic stages; Species identification; Vectors
Doxycycline has been proposed for the treatment of malnourished children in developing countries, and its use has been associated with weight gain in healthy volunteers. No previous studies have assessed abnormal weight gain as a putative side effect of long-term doxycycline treatment; thus, the objective of the present study was to characterize this phenomenon. We also analyzed the role of the gut microbiota in this effect. We assessed changes in the body mass index in Q fever endocarditis patients treated with doxycycline and hydroxychloroquine and healthy individuals with no antibiotic treatment. Abnormal weight gain was defined as a gain in weight above that of the controls. The fecal samples were examined using molecular assays for Methanobrevibacter smithii, Bacteroidetes, Firmicutes, Escherichia coli, Lactobacillus, Lactobacillus reuteri, and total bacterial concentrations. We examined 82 patients, including 48 patients with Q fever endocarditis and 34 controls. Approximately 23% of the treated patients showed abnormal weight gain (P = 0.001). Patients treated with doxycycline and hydroxychloroquine presented significantly lower concentrations of Bacteroidetes (P = 0.002), Firmicutes (P = 0.01), and Lactobacillus (P = 0.02). The linear regression analysis revealed that the duration of treatment was significantly associated with a decrease in Bacteroidetes (P = 0.0001), Firmicutes (P = 0.002), and total bacteria (P < 0.00001). Abnormal weight gain is a side effect of long-term doxycycline and hydroxychloroquine treatment. Gut microbiota modifications at the phylum level could play an instrumental role in this effect. We highlight the need for specific nutritional care in patients undergoing long-term antibiotic treatment, particularly treatment involving the use of doxycycline.
Coxiella burnetii Cb196, with a 2,006,415-bp genome, is a strain isolated from a 45-year-old man in Saudi Arabia with endocarditis. It belongs to the genotype MST51, which was detected for the first time only in this country. Cb196 shows more similarity to C. burnetii CbuK_Q154, belonging to genotype 8, which was phylogenetically close to MST51.
Kingella kingae strain KK247 was isolated from an adult Israeli patient with endocarditis. It belongs to pulsed-field gel electrophoresis clone A, has a 2,113,021-bp genome, a 15,507-bp plasmid that carries genes encoding β-lactamases, and possesses 45 transposases, compared to the 5 detected in other K. kingae strains.
Wild apes are considered to be the most serious reservoir and source of zoonoses. However, little data are available about the gut microbiota and pathogenic bacteria in gorillas. For this propose, a total of 48 fecal samples obtained from 21 Gorilla gorilla gorilla individuals (as revealed via microsatellite analysis) were screened for human bacterial pathogens using culturomics and molecular techniques. By applying culturomics to one index gorilla and using specific media supplemented by plants, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic pathogens were isolated, including 8 frequently associated with human diseases; Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. The genus Treponema accounted for 27.4% of the total reads identified at the genus level via 454 pyrosequencing. Using specific real-time PCR on 48 gorilla fecal samples, in addition to classical human pathogens, we also observed the fastidious bacteria Bartonella spp. Borrelia spp., Coxiella burnetii and Tropheryma whipplei in the gorilla population. We estimated that the prevalence of these pathogens vary between 4.76% and 85.7%. Therefore, gorillas share many bacterial pathogens with humans suggesting that they could be a reservoir for their emergence.
The draft genome sequence of Borrelia crocidurae strain 03-02, a blood isolate from a febrile Senegalese patient, comprises a 920,021-bp linear chromosome (27.7% G+C content), 32 tRNAs, 818 open reading frames, and one cluster of regularly interspaced short palindromic repeats. Its genotype differs from that of the Achema reference strain.
Rickettsia hoogstraalii is a tick-associated member of the spotted fever group rickettsiae that is geographically widely distributed. We report here the draft genome of R. hoogstraalii strain CroaticaT (=DSM 22243 = UTMB 00003), which was isolated from Haemaphysalis sulcata ticks collected in Croatia.
Coprolites are fossilized fecal material that can reveal information about ancient intestinal and environmental microbiota. Viral metagenomics has allowed systematic characterization of viral diversity in environmental and human-associated specimens, but little is known about the viral diversity in fossil remains. Here, we analyzed the viral community of a 14th-century coprolite from a closed barrel in a Middle Ages site in Belgium using electron microscopy and metagenomics. Viruses that infect eukaryotes, bacteria, and archaea were detected, and we confirmed the presence of some of them by ad hoc suicide PCR. The coprolite DNA viral metagenome was dominated by sequences showing homologies to phages commonly found in modern stools and soil. Although their phylogenetic compositions differed, the metabolic functions of the viral communities have remained conserved across centuries. Antibiotic resistance was one of the reconstructed metabolic functions detected.
The most common pathogens detected were coronaviruses, rhinoviruses, influenza viruses, and Streptococcus pneumoniae.
Pilgrims returning from the Hajj might contribute to international spreading of respiratory pathogens. Nasal and throat swab specimens were obtained from 129 pilgrims in 2013 before they departed from France and before they left Saudi Arabia, and tested by PCR for respiratory viruses and bacteria. Overall, 21.5% and 38.8% of pre-Hajj and post-Hajj specimens, respectively, were positive for ≥1 virus (p = 0.003). One third (29.8%) of the participants acquired ≥1 virus, particularly rhinovirus (14.0%), coronavirus E229 (12.4%), and influenza A(H3N2) virus (6.2%) while in Saudi Arabia. None of the participants were positive for the Middle East respiratory syndrome coronavirus. In addition, 50.0% and 62.0% of pre-Hajj and post-Hajj specimens, respectively, were positive for Streptococcus pneumoniae (p = 0.053). One third (36.3%) of the participants had acquired S. pneumoniae during their stay. Our results confirm high acquisition rates of rhinovirus and S. pneumoniae in pilgrims and highlight the acquisition of coronavirus E229.
bacteria; viruses; cohort study; Hajj; respiratory tract infections; pilgrims; Saudi Arabia
The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia.
The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia.
To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage.
Measurements and Primary Results
SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality.
These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.
We present the draft genome sequence of Legionella massiliensis strain LegAT, recovered from a cooling tower water sample, using an amoebal coculture procedure. The strain described here is composed of 4,387,007 bp, with a G+C content of 41.19%, and its genome has 3,767 protein-coding genes and 60 predicted RNA genes.
Little is known about the presence/absence and prevalence of Rickettsia spp, Bartonella spp. and Yersinia pestis in domestic and urban flea populations in tropical and subtropical African countries.
Fleas collected in Benin, the United Republic of Tanzania and the Democratic Republic of the Congo were investigated for the presence and identity of Rickettsia spp., Bartonella spp. and Yersinia pestis using two qPCR systems or qPCR and standard PCR. In Xenopsylla cheopis fleas collected from Cotonou (Benin), Rickettsia typhi was detected in 1% (2/199), and an uncultured Bartonella sp. was detected in 34.7% (69/199). In the Lushoto district (United Republic of Tanzania), R. typhi DNA was detected in 10% (2/20) of Xenopsylla brasiliensis, and Rickettsia felis was detected in 65% (13/20) of Ctenocephalides felis strongylus, 71.4% (5/7) of Ctenocephalides canis and 25% (5/20) of Ctenophthalmus calceatus calceatus. In the Democratic Republic of the Congo, R. felis was detected in 56.5% (13/23) of Ct. f. felis from Kinshasa, in 26.3% (10/38) of Ct. f. felis and 9% (1/11) of Leptopsylla aethiopica aethiopica from Ituri district and in 19.2% (5/26) of Ct. f. strongylus and 4.7% (1/21) of Echidnophaga gallinacea. Bartonella sp. was also detected in 36.3% (4/11) of L. a. aethiopica. Finally, in Ituri, Y. pestis DNA was detected in 3.8% (1/26) of Ct. f. strongylus and 10% (3/30) of Pulex irritans from the villages of Wanyale and Zaa.
Most flea-borne infections are neglected diseases which should be monitored systematically in domestic rural and urban human populations to assess their epidemiological and clinical relevance. Finally, the presence of Y. pestis DNA in fleas captured in households was unexpected and raises a series of questions regarding the role of free fleas in the transmission of plague in rural Africa, especially in remote areas where the flea density in houses is high.
Fleas are associated with many bacterial diseases such as rickettsioses, bartonelloses and plague. These diseases may be severe, and little is known about their prevalence. Accordingly, we believe that our data shed light on the problem of unexplained fevers in tropical and subtropical African areas. Using molecular tools, we surveyed and studied selected flea-borne agents, namely Rickettsia spp. (R. felis and R. typhi), Bartonella spp. and Y. pestis, in fleas collected in Ituri (Linga and Rethy health zone) and Kinshasa in the Democratic Republic of the Congo, the Lushoto district in the United Republic of Tanzania and in Cotonou in Benin. We found that these bacteria are present in the studied regions. R. typhi and an unidentified Bartonella sp. were detected in fleas from Cotonou (Benin). R. felis and R. typhi were also detected in the Lushoto district (United Republic of Tanzania). Finally, we detected R. felis, Bartonella sp. and Y. pestis in the Democratic Republic of the Congo. As these emerging zoonotic diseases have a global distribution and affect public health, the implementation of vector control strategies is urgent.
Necropsobacter is a recently described genus that contains a single species, N. rosorum, and belongs to the family Pasteurellaceae. Here, we present the draft genome of N. rosorum strain P709T, which is the first genome sequence from this species.
Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convex side of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open-eight’ conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.
Lymph node enlargement is a common medical problem, and in a large number of patients, the causes of lymphadenopathy remain undiagnosed. We report a thorough microbiological analysis of 1,688 lymph node biopsy specimens collected in our bartonellosis reference center. We studied lymph node biopsy samples from patients with suspected regional infectious lymph node enlargement from January 2008 to December 2012. To evaluate a useful strategy for the diagnosis of infectious lymphadenitis, specimens were cultured and subjected to molecular assays. Histologic analysis was done when possible. A total of 642 (38%) biopsy specimens were infected with a bacterial agent, and quantitative PCR (qPCR) was significantly better than 16S rRNA gene PCR (rrs) for the detection of Bartonella henselae (P = 0.05), Mycobacterium tuberculosis (P = 0.05), and Mycobacterium avium (P = 0.007). Molecular assays were significantly better than bacterial cultures for the diagnosis of Francisella tularensis (P = 0.017) but were less effective for detecting M. tuberculosis (P = 0.004) and M. avium (P = 0.001). Histologic analysis was done for 412 lymph nodes, and 20% of these were compatible with an infectious lymphadenitis, whereas a neoplasm was found in 29% of these lymph nodes. M. tuberculosis was detected significantly more in female than in male patients (P = 0.01), and patients with cat scratch disease (CSD) were younger than patients with M. tuberculosis, Tropheryma whipplei, and F. tularensis. Negative rrs PCR does not exclude the diagnosis of infectious lymphadenitis. Histologic analysis of lymph node biopsy specimens is critical, as a diagnosis of infectious lymphadenitis does not preclude other concurrent diseases.
Osteoarticular infection is an uncommon presentation of Q fever. Positron emission tomography (PET) scanning is a valuable tool for the diagnosis of Coxiella burnetii graft prosthesis infection and endocarditis. Our objective was to test a series of culture-negative osteoarticular samples using molecular assays for Coxiella burnetii. We tested for C. burnetii by molecular assays targeting the IS1111 and the IS30A spacer regions, using culture-negative osteoarticular samples obtained in our laboratory between January 2011and December 2012. We examine a total of 1,410 osteoarticular samples, and we observed two cases of arthritis and subacromial bursitis caused by C. burnetii. The infections were localized using PET scanning, and the diagnosis was confirmed through serology. For one, a C. burnetii strain with a multispacer sequence type 8 genotype was isolated from synovial fluid culture. Q fever articular infections could be undiagnosed because of the long evolution of articular attack, and patients with high antibody titers against C. burnetii should be tested using PET scanning to localize the site of infection.