IVDR: Analysis of the Social, Economic, and Practical Consequences of the Application of an Ordinance of the In Vitro Diagnostic Ordinance in Switzerland.

IVDR regulation represents a major challenge for diagnostic microbiology laboratories. IVDR complicates a broad range of aspects and poses a risk given the high diversity of pathogens (including rare but highly virulent microbes) and the large variety of samples submitted for analysis. The regular emergence of new pathogens (including Echovirus E-11, Adenovirus 41, Monkeypox virus, Alongshan virus, and Enterovirus D68, as recent examples in Europe in the post SARS-CoV-2 era) is another factor that makes IVDR regulation risky, because its detrimental effect on production of in-house tests will negatively impact knowledge and expertise in the development of new diagnostic tests. Moreover, such regulations negatively impact the availability of diagnostic tests, especially for neglected pathogens, and has a detrimental effect on the overall costs of the tests. The increased regulatory burden of IVDR may thereby pose an important risk for public health. Taken together, it will have a negative impact on the financial balance of diagnostic microbiology laboratories (especially small ones). The already-high standards of quality management of all ISO-accredited and Swissmedic-authorized laboratories render IVDR law of little value, at least in Switzerland, while tremendously increasing the regulatory burden and associated costs. Eventually, patients will need to pay for diagnostic assays outside of the framework of their insurance in order to obtain a proper diagnostic assessment, which may result in social inequity. Thus, based on the risk assessment outlined above, the coordinated commission for clinical microbiology proposes adjusting the IvDO ordinance by (i) introducing an obligation to be ISO 15189 accredited and (ii) not implementing the IvDO 2028 milestone.

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

Multifocal lymphadenopathies with polyclonal reactions primed after EBV infection in a mRNA-1273 vaccine recipient.

We report a case of recurrent tender, multifocal lymphadenopathies associated with B-symptoms, clinically mimicking lymphoma in a mRNA-1273 vaccine recipient after a recent Epstein-Barr virus (EBV) infection. In the lymph node biopsy, monocytoid B-cell hyperplasia, TH2 (GATA3+) predominance, and hyperplasia of interferon-gamma-producing plasmacytoid dendritic cells were observed along with sustained neutralising antibody production against SARS-CoV-2 wild-type and five variants. High titres of anti-S antibodies and neutralising antibodies were observed, excepted for variant B.1.529** (omicron) and B.1.351** (beta), due to several mutations in the spike protein, including the E484K mutation. We postulated that EBV acted as an immunological enhancer with the mRNA-1273 vaccine, inducing a sustained inflammatory response over several weeks. However, the polyclonal nature of the lymphadenopathy with polytypic plasmacytosis and pseudo-tumoural reaction cell hyperplasia were associated with failure to mount acute phase responses.

Unexpected associations between respiratory viruses and bacteria with Pulmonary Function Testing in children suffering from Cystic Fibrosis (MUCOVIB study).

BACKGROUND: Various bacterial and viral assemblages composing Cystic Fibrosis (CF) lung microbiota contribute to long-term lung function decline over time. Yet, the impact of individual microorganisms on pulmonary functions remains uncertain in children with CF. METHODS: As part of the 'Mucoviscidosis, respiratory VIruses, intracellular Bacteria and fastidious organisms'' project, children with CF were longitudinally followed in a Swiss multicentric study. Respiratory samples included mainly throat swabs and sputa samples for bacterial culture and 16S rRNA metagenomics and nasopharyngeal swabs for respiratory virus detection by molecular assays. Percentage of predicted Forced Expiratory Volume in one second (FEV1%) and Lung Clearance Index (LCI) were recorded. RESULTS: Sixty-one children, of whom 20 (32.8%) presented with at least one pulmonary exacerbation, were included. Almost half of the 363 nasopharyngeal swabs tested by RT-PCR were positive for a respiratory virus, mainly rhinovirus (26.5%). From linear mixed-effects regression models, P. aeruginosa (-11.35, 95%CI [-17.90; -4.80], p = 0.001) was significantly associated with a decreased FEV1%, whereas rhinovirus was associated with a significantly higher FEV1% (+4.24 95%CI [1.67; 6.81], p = 0.001). Compared to conventional culture, 16S rRNA metagenomics showed a sensitivity and specificity of 80.0% and 85.4%, respectively for detection of typical CF pathogens. However, metagenomics detected a bacteria almost twice more often than culture. CONCLUSIONS: As expected, P. aeruginosa impacted negatively on FEV1% while rhinovirus was surprisingly associated with better FEV1%. Culture-free assays identifie significantly more pathogens than standard culture, with disputable clinical correlation.

Decreased Fetal Movements: A Sign of Placental SARS-CoV-2 Infection with Perinatal Brain Injury.

Neonatal COVID-19 is rare and mainly results from postnatal transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, can infect the placenta and compromise its function. We present two cases of decreased fetal movements and abnormal fetal heart rhythm 5 days after mild maternal COVID-19, requiring emergency caesarean section at 29 + 3 and 32 + 1 weeks of gestation, and leading to brain injury. Placental examination revealed extensive and multifocal chronic intervillositis, with intense cytoplasmic positivity for SARS-CoV-2 spike antibody and SARS-CoV-2 detection by RT-qPCR. Vertical transmission was confirmed in one case, and both neonates developed extensive cystic peri-ventricular leukomalacia.

Chlamydia pneumoniae and Mycoplasma pneumoniae in children with cystic fibrosis: impact on bacterial respiratory microbiota diversity.

OBJECTIVES: The contribution of intracellular and fastidious bacteria in Cystic fibrosis (CF) pulmonary exacerbations, and progressive lung function decline remains unknown. This project aimed to explore their impact on bacterial microbiota diversity over time in CF children. METHODS: Sixty-one children enrolled in the MUCOVIB multicentre prospective cohort provided 746 samples, mostly nasopharyngeal swabs, throat swabs and sputa which were analysed using culture, specific real-time qPCRs and 16S rRNA amplicon metagenomics. RESULTS: Chlamydia pneumoniae (n = 3) and Mycoplasma pneumoniae (n = 1) were prospectively documented in 6.6% of CF children. Microbiota alpha-diversity in children with a documented C. pneumoniae was highly variable, similarly to children infected with Staphylococcus aureus or Pseudomonas aeruginosa. The transition from routine follow-up visits to pulmonary exacerbation (n = 17) yielded variable changes in diversity indexes with some extreme loss of diversity. CONCLUSIONS: The high rate of C. pneumoniae detection supports the need for regular screenings in CF patients. A minor impact of C. pneumoniae on the microbial community structure was documented. Although detected in a single patient, M. pneumoniae should also be considered as a possible aetiology of lung infection in CF subjects.

Transcriptional Landscape of Waddlia chondrophila Aberrant Bodies Induced by Iron Starvation.

Chronic infections caused by obligate intracellular bacteria belonging to the Chlamydiales order are related to the formation of persistent developmental forms called aberrant bodies (ABs), which undergo DNA replication without cell division. These enlarged bacteria develop and persist upon exposure to different stressful conditions such as ß-lactam antibiotics, iron deprivation and interferon-γ. However, the mechanisms behind ABs biogenesis remain uncharted. Using an RNA-sequencing approach, we compared the transcriptional profile of ABs induced by iron starvation to untreated bacteria in the Chlamydia-related species Waddliachondrophila, a potential agent of abortion in ruminants and miscarriage in humans. Consistent with the growth arrest observed following iron depletion, our results indicate a significant reduction in the expression of genes related to energy production, carbohydrate and amino acid metabolism and cell wall/envelope biogenesis, compared to untreated, actively replicating bacteria. Conversely, three putative toxin-antitoxin modules were among the most up-regulated genes upon iron starvation, suggesting that their activation might be involved in growth arrest in adverse conditions, an uncommon feature in obligate intracellular bacteria. Our work represents the first complete transcriptomic profile of a Chlamydia-related species in stressful conditions and sets the grounds for further investigations on the mechanisms underlying chlamydial persistence.

[Myases from here and elsewhere†: pseudo-furonculosis and Ignatzschineria larvae bacteremia]. / Myiases d'ici et d'ailleurs†: pseudo-furonculose et bactériémie à Ignatzschineria larvae.

Myiasis is an infestation by maggots. In humans, it predominates in regions with low socio-economic development. We report on two cases of myiasis acquired during a tropical travel and in Switzerland, respectively. The first one presented as a furunculous-like disease due to the invasion of subcutaneous tissues by Cordylobia sp. larvae. The second corresponded to a chronic wound infestation that resulted in a rarely reported bacteremia due to Ignatzschineria larvae, a commensal bacteria of maggots' digestive tract. Surgery was necessary in both cases, mainly for psychological reasons in the first case. Both the entomologist and molecular biology were instrumental for treatment decisions.

La myiase est une infestation par des larves de mouches. Chez l'homme, elle prédomine dans les régions à faible niveau socio-économique. Nous rapportons ici deux cas de myiase, l'un acquis lors d'un voyage sous les tropiques et l'autre autochtone : une myiase furonculaire due à la pénétration d'une larve de diptère dans la peau, en l'occurrence Cordylobia sp. ; et une myiase de plaie survenue par ponte de mouches dans des tissus nécrotiques, avec une exceptionnelle bactériémie secondaire, due à une bactérie commensale du tractus digestif de ces larves, Ignatzschineria larvae. Dans les deux situations, la chirurgie a été nécessaire, pour une indication surtout d'ordre psychologique dans la première. Dans les deux cas, l'apport de l'entomologiste et de la biologie moléculaire a été déterminant dans la décision thérapeutique.

Assessing the efficacy and impact of a personalised smoking cessation intervention among type 2 diabetic smokers: study protocol for an open-label randomised controlled trial (DISCGO-RCT).

INTRODUCTION: Few studies have assessed the efficacy of smoking cessation interventions in individuals with type 2 diabetes, but interventions adapted to the specific needs of this population are warranted. The aim of this study is to assess the efficacy of a smoking cessation intervention in a population of smokers with type 2 diabetes and to measure the metabolic impact of smoking cessation. METHODS AND ANALYSIS: The study is an open-label, randomised control trial. Participants recruited from a sanitary region of Switzerland will be randomly allocated to either the intervention or the control arm. The intervention group will have four individual counselling sessions over 12 weeks. Trained research nurses will conduct the behavioural intervention, using motivational interviews and addressing diabetes and gender specificities. The control group will have one short counselling session at baseline and will be given written information on smoking cessation. Both groups will have a follow-up visit at 26 and 52 weeks. Demographic and medical data will be collected at baseline and follow-up, along with blood and urine samples. The primary study outcome is continuous smoking abstinence validated by expired-air carbon monoxide from week 12 to week 52. Secondary study outcomes are continuous and 7-day point prevalence smoking abstinence at 12 and 26 weeks; change in motivation to quit and cigarette consumption; and change in glycosylated haemoglobin levels, body weight, waist circumference and renal function after smoking cessation. In a subsample of 80 participants, change in stool microbiota from baseline will be measured at 3, 8 and 26 weeks after smoking cessation. ETHICS AND DISSEMINATION: Ethical approval has been obtained by the competent ethics committee (Commission cantonale d'éthique de la recherche sur l'être humain, CER-VD 2017-00812). The results of the study will be disseminated through publications in peer-reviewed journals and conference presentations. TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov NCT03426423 and SNCTP000002762; Pre-results.

CAPNOCYTOPHAGA CANIMORSUS ENDOPHTHALMITIS AFTER CATARACT SURGERY LINKED TO SALIVARY DOG-TO-HUMAN TRANSMISSION.

PURPOSE: To describe a case of acute postoperative bacterial endophthalmitis because of Capnocytophaga canimorsus after cataract surgery, with probable contamination through salivary droplets of dog two days after the procedure. METHODS: An 83-year-old woman who underwent uncomplicated cataract extraction with intraocular lens implantation, presented 12 days later with acute pain, redness, and vision loss in her left eye. Visual acuity was hand motion and clinical findings suggested the diagnosis of acute postoperative endophthalmitis. The patient underwent diagnostic vitrectomy, intravitreal ceftazidime/vancomycin injection and received oral moxifloxacin (400 mg/day). Two days later, she underwent complete pars-plana vitrectomy because of the absence of clinical improvement. Vitreous samples showed gram-negative bacterium on direct examination but cultures remained sterile, which prompted the realization of a broad-range bacterial polymerase chain reaction analysis. RESULTS: Polymerase chain reaction on the vitreous sample detected C. canimorsus, a fastidious gram-negative bacterium of the oral canine flora. When asked for recent contact with dogs, the patient reported having proceeded to an intensive tooth care session for her dog at postoperative Day 2. Intravenous ceftriaxone (2 g/day) was added to the treatment. Anterior and posterior segment inflammation slowly resolved, and final visual acuity was 20/160. CONCLUSION: Although very rare, this complication suggests that patients undergoing ocular surgery should avoid contact with salivary secretions of pets during the early postoperative period. Diagnostic broad-range bacterial polymerase chain reaction is useful to detect unconventional or slow-growing agents in vitreous samples.

Phylogenomics reveal that Mycobacterium kansasii subtypes are species-level lineages. Description of Mycobacterium pseudokansasii sp. nov., Mycobacterium innocens sp. nov. and Mycobacterium attenuatum sp. nov.

Among the species Mycobacterium kansasii, seven subtypes have been previously reported based on the PCR and the restriction fragment length polymorphism of the gene hsp65. Here, we used whole-genome sequencing to refine M. kansasii taxonomy and correct multiple inconsistencies. Average nucleotide identity (ANI) values between M. kansasii subtypes ranged from 88.4 to 94.2 %, lower than the accepted 95-96 % cut-off for species delineation. In addition, Mycobacterium gastri was closer to the M. kansasii subtypes 1, 2, 3, 4 and 5 than M. kansasii subtype 6. The recently described species Mycobacterium persicum shared 99.77 % ANI with M. kansasii subtype 2. Consistent with the ANI results, the digital DNA-DNA hybridization value was below the 70 % threshold for species delineation between subtypes and above it within subtypes as well as between subtype 2 and M. persicum. Furthermore, core-genome phylogeny confirmed the current M. kansasii species to be polyphyletic. Hence, we propose (i) Mycobacterium pseudokansasii sp. nov., replacing subtype 3, with the type strain MK142T(=CCUG 72128T=DSM 107152T), (ii) Mycobacterium innocens sp. nov., replacing subtype 5, with the type strain MK13T (=CCUG 72126T=DSM 107161T), and (iii) Mycobacterium attenuatum sp. nov., replacing subtype 6, with the type strain MK41T(=CCUG 72127T=DSM 107153T). Subtype 4 represents a new species-level lineage based on the genomic data but no strain was available. No genome sequence or strain was available for subtype 7. The proposed nomenclature will facilitate the identification of the most pathogenic subtype 1 as M. kansasii by clinicians while the new species names suggest the attenuated pathogenicity of the other subtypes.

Molecular diagnosis and enrichment culture identified a septic pseudoarthrosis due to an infection with Erysipelatoclostridium ramosum.

We describe here a rare case of septic pseudarthrosis due to Erysipelatoclostridium ramosum in a female young patient. The patient, currently in remission from Ewing's sarcoma treated by a bone resection and allograft combined with chemotherapy, suffered from a chronic femoral pseudarthrosis in a context of bone insufficiency and graft resorption. A broad range 16S PCR followed by sequencing, as well as an enrichment culture of a bone biopsy revealed the presence of E. ramosum, an anaerobic firmicute with a low Gram-positive affinity staining and low GC content, that was further characterized by whole genome sequencing (WGS).

Antibodies to Chlamydia trachomatis and reproductive health issues in women with SLE: a case-control study.

BACKGROUND: SLE is an autoimmune condition affecting predominantly women. Little is known regarding Chlamydia trachomatis infection in women with SLE, which may drive autoimmunity and contribute to obstetrical and vascular complications. METHODS: This single-centre, case-control study set primary endpoint in the comparative seropositivity rate to C. trachomatis major outer membrane protein (MOMP) and chlamydial heat-shock protein-60 (cHSP60) in age-matched subjects. The secondary endpoints were obstetrical outcomes, cardiovascular events and results from screening procedures for cervical cancer. RESULTS: Eighty-four women with SLE and 50 age-matched controls were included. Seropositivity to C. trachomatis did not differ significantly between groups (10% of cases positive for anti-MOMP vs 12% of controls; 43% of cases positive for anti-cHSP60 vs 32% of controls). Women with SLE were more often of non-Caucasian ethnicity and had lower educational level. They relied less frequently on oral contraception and resorted more frequently to elective pregnancy termination. Pre-eclampsia and ectopic pregnancy occurred only in SLE. Women with SLE also experienced more cardiovascular events. In SLE, antibodies to cHSP60 were associated with a history of pericarditis and abnormal screening tests for cervical cancer. Antibody titres to C. trachomatis were not associated with disease activity or SLE treatment, nor were there associations with other gynaecological, obstetrical or vascular outcomes. CONCLUSION: Prevalence of antibodies to C. trachomatis was not increased in women with SLE. No significant association was found between these antibodies and obstetrical or cardiovascular complications.

Limited added value of fungal ITS amplicon sequencing in the study of bovine abortion.

Bovine mycotic abortion is sporadic and caused by different ubiquitous and opportunistic fungi. Recently, a broad spectrum of bacterial opportunists involved in bovine abortion was revealed by 16S rRNA gene amplicon sequencing. We hypothesized that fungal organisms potentially involved in bovine abortion also might remain undetected by conventional culture. In this retrospective study, we therefore applied fungal internal transcribed spacer 2 (ITS2) region amplicon sequencing to 74 cases of bovine abortion submitted to our diagnostic service. The investigation was complemented by fungal culture and, retrospectively, by data from bacteriological, virological and parasitological analyses and histopathological examination of placentas. Fungal DNA was found in both the placentas and abomasal contents, with 92 fungal genera identified. In 18 cases, >75% of the reads belonged to one specific fungal genus: Candida (n = 7), Malassezia (n = 4), Cryptococcus (n = 3), unidentified Capnodiales (n = 3), Actinomucor (n = 1), Cystofilobasidium (n = 1), Penicillium (n = 1), Verticillum (n = 1) and Zymoseptoria (n = 1) with one case harboring two different genera. By culture, in contrast, fungal agents were detected in only 6 cases. Inflammatory and/or necrotizing lesions were found in 27/40 histologically assessed placentas. However, no lesion-associated fungal structures were detected in HE- and PAS-stained specimens. Complementary data revealed the presence of one or more non-fungal possible abortifacient: Chlamydiales, Coxiella burnetii, Leptospira spp., Campylobacter fetus subsp. fetus, Streptococcus uberis, Escherichia coli, Streptococcus pluranimalium, Bacillus licheniformis, Campylobacter fetus subsp. fetus, Serratia marcescens, Trueperella pyogenes, Schmallenbergvirus, Neospora caninum. The mycobiota revealed by sequencing did not differ between cases with or without a possible infectious etiology. Our study suggests that amplicon sequencing of the ITS2 region from DNA isolated from bovine abortion does not provide additional information or new insight into mycotic abortion and without complementary analyses may easily lead to a false interpretation of the role of fungal organisms in bovine abortion.

Discovery and Characterization of Mycobacterium basiliense sp. nov., a Nontuberculous Mycobacterium Isolated From Human Lungs.

Bacteria belonging to the genus Mycobacterium are predominantly responsible for pulmonary diseases; most notably Mycobacterium tuberculosis causes granulomatous pulmonary infections. Here we describe a novel slow growing mycobacterial species isolated from respiratory samples from five patients, four with underlying pulmonary disease. The isolates were characterized by biochemical and molecular techniques, including whole genome sequencing. Biochemical characteristics generally match those of M. marinum and M. ulcerans; however, the most striking difference of the new species is its ability to grow at 37°C. The new species was found to grow in human macrophages, but not amoebae, suggesting a pathogenic rather than an environmental lifestyle. Phylogenetic analysis reveals a deep-rooting relationship to M. marinum and M. ulcerans. A complete genome sequence was obtained through combining short and long-read sequencing, providing a genome of 5.6 Mb. The genome appears to be highly intact, syntenic with that of M. marinum, with very few insertion sequences. A vast array of virulence factors includes 283 PE/PPE surface-associated proteins, making up 10% of the coding capacity, and 22 non-ribosomal peptide synthase clusters. A comparison of six clinical isolates from the five patients shows that they differ by up to two single nucleotide polymorphisms, suggesting a common source of infection. Our findings are in accordance with the recognition of a new taxonomic entity. We propose the name M. basiliense, as all isolates were found in patients from the Basel area of Switzerland.

Impact of HPV vaccination with Gardasil® in Switzerland.

BACKGROUND: Gardasil®, a quadrivalent vaccine targeting low-risk (6, 11) and high-risk (16, 18) human papillomaviruses (HPV), has been offered to 11-14 year-old schoolgirls in Switzerland since 2008. To evaluate its success and its potential impact on cervical cancer screening, HPV genotypes were examined in 18-year-old girls five years later (sub-study 1) and in outpatients participating to cervical cancer screening before and after vaccine implementation (sub-study 2). METHODS: For sub-study 1, 3726 females aged 18 in 2013 were invited to fill a questionnaire on personal demographics and HPV risk factors and to provide a self-collected cervicovaginal sample for HPV genotyping and Chlamydia trachomatis PCR. Personal data were evaluated by univariable and multivariable statistics. In sub-study 2, the proportion of the vaccine-type HPV among anogenital HPV was examined with archived genotyping data of 8039 outpatients participating to cervical cancer screening from 1999 till 2015. The yearly evolution of this proportion was evaluated by segmented logistic regression. RESULTS: 690 (18.5%) women participated to sub-study 1 and 327 (8.8%) provided a self-collected sample. Prevalence of Chlamydia trachomatis (4.6%) and demographics confirmed that the subjects were representative of sexually-active Swiss young women. Vaccine (five-year coverage: 77.5%) was preferentially accepted by contraceptive-pill users (P = 0.001) and samples were mainly provided by sexually-active subjects (P < 0.001). The proportion (4%) of the vaccine-type HPV in this population was lower than in sub-study 2 outpatients (n = 849, <26 years old) in the pre-vaccine era (25.7%). The proportion of the high-risk vaccine-type HPV decreased significantly (59%, P = 0.0048) in the outpatients during the post-vaccine era, yet this decrease was restricted to those aged less than 26 years (n = 673, P < 0.0001). CONCLUSIONS: The low proportion of vaccine-type HPV in 18-year-old females and its rapid decrease in young women participating to cervical cancer screening extend the success of HPV vaccination to Switzerland. Our data suggest that cervical cancer screening is now entering a stage of reduced proportion of HPV16 and/or 18 in samples reported positive by cytology. In view of the high likelihood of reduced clinical specificity of cytology, primary screening modalities involving HPV testing and cytology should now be re-evaluated in Switzerland.

The Anticancer Peptide TAT-RasGAP317-326 Exerts Broad Antimicrobial Activity.

Antibiotic resistance has become a major health issue. Nosocomial infections and the prevalence of resistant pathogenic bacterial strains are rising steadily. Therefore, there is an urgent need to develop new classes of antibiotics effective on multi-resistant nosocomial pathogenic bacteria. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, induces cancer cell death, inhibits metastatic progression, and sensitizes tumor cells to various anti-cancer treatments in vitro and in vivo. We here report that TAT-RasGAP317-326 also possesses antimicrobial activity. In vitro, TAT-RasGAP317-326, but not mutated or truncated forms of the peptide, efficiently killed a series of bacteria including Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa. In vivo experiments revealed that TAT-RasGAP317-326 protects mice from lethal E. coli-induced peritonitis if administrated locally at the onset of infection. However, the protective effect was lost when treatment was delayed, likely due to rapid clearance and inadequate biodistribution of the peptide. Peptide modifications might overcome these shortcomings to increase the in vivo efficacy of the compound in the context of the currently limited antimicrobial options.

Culture-independent genomics of a novel chlamydial pathogen of fish provides new insight into host-specific adaptations utilized by these intracellular bacteria.

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

Messages from the first International Conference on Clinical Metagenomics (ICCMg).

Metagenomics is recently entering in the clinical microbiology and an increasing number of diagnostic laboratories are now proposing the sequencing & annotation of bacterial genomes and/or the analysis of clinical samples by direct or PCR-based metagenomics with short time to results. In this context, the first International Conference on Clinical Metagenomics (ICCMg) was held in Geneva in October 2016 and several key aspects have been discussed including: i) the need for improved resolution, ii) the importance of interpretation given the common occurrence of sequence contaminants, iii) the need for improved bioinformatic pipelines, iv) the bottleneck of DNA extraction, v) the importance of gold standards, vi) the need to further reduce time to results, vii) how to improve data sharing, viii) the applications of bacterial genomics and clinical metagenomics in better adapting therapeutics and ix) the impact of metagenomics and new sequencing technologies in discovering new microbes. Further efforts in term of reduced turnaround time, improved quality and lower costs are however warranted to fully translate metagenomics in clinical applications.

Simkania negevensis may produce long-lasting infections in human pneumocytes and endometrial cells.

Simkania negevensis is a novel Chlamydia-related bacterium and the founding member of the Simkaniaceae family within the Chlamydiales order. Little is known about the biology and pathogenesis of this bacterium. So far, S. negevensis has been considered as an amoebal symbiont, but its natural host remains unknown. Moreover, evidence of human exposition has been reported worldwide and an association with pneumonia and bronchiolitis is suspected. Here, we evaluated the ability of S. negevensis to replicate in potential environmental reservoirs, namely amoebae and arthropods, as well as in mammalian cells (Vero cells, pneumocytes and endometrial cells) and further evaluated the characteristics of its replicative vacuole. We demonstrated that S. negevensis efficiently replicates in all cell lines tested, with the shortest doubling time and an increased adhesion observed in pneumocytes. Our work highlights the specificities of the Simkania-containing vacuole compared to other Chlamydiales; contrarily to Chlamydia trachomatis, S. negevensis does not disrupt the Golgi apparatus. Importantly, our work suggests that S. negevensis infection is associated with few cytopathic effects and might persist for a prolonged time in infected cells. Further evaluation of its implication in human diseases is required; an implication in chronic or subacute respiratory infections might be suspected.