The genomic and phylogenetic analysis of Marseillevirus cajuinensis raises questions about the evolution of Marseilleviridae lineages and their taxonomical organization.

Marseilleviruses (MsV) are a group of viruses that compose the Marseilleviridae family within the Nucleocytoviricota phylum. They have been found in different samples, mainly in freshwater. MsV are classically organized into five phylogenetic lineages (A/B/C/D/E), but the current taxonomy does not fully represent all the diversity of the MsV lineages. Here, we describe a novel strain isolated from a Brazilian saltwater sample named Marseillevirus cajuinensis. Based on genomics and phylogenetic analyses, M. cajuinensis exhibits a 380,653-bp genome that encodes 515 open reading frames. Additionally, M. cajuinensis encodes a transfer RNA, a feature that is rarely described for Marseilleviridae. Phylogeny suggests that M. cajuinensis forms a divergent branch within the MsV lineage A. Furthermore, our analysis suggests that the common ancestor for the five classical lineages of MsV diversified into three major groups. The organization of MsV into three main groups is reinforced by a comprehensive analysis of clusters of orthologous groups, sequence identities, and evolutionary distances considering several MsV isolates. Taken together, our results highlight the importance of discovering new viruses to expand the knowledge about known viruses that belong to the same lineages or families. This work proposes a new perspective on the Marseilleviridae lineages organization that could be helpful to a future update in the taxonomy of the Marseilleviridae family. IMPORTANCE: Marseilleviridae is a family of viruses whose members were mostly isolated from freshwater samples. In this work, we describe the first Marseillevirus isolated from saltwater samples, which we called Marseillevirus cajuinensis. Most of M. cajuinensis genomic features are comparable to other Marseilleviridae members, such as its high number of unknown proteins. On the other hand, M. cajuinensis encodes a transfer RNA, which is a gene category involved in protein translation that is rarely described in this viral family. Additionally, our phylogenetic analyses suggested the existence of, at least, three major Marseilleviridae groups. These observations provide a new perspective on Marseilleviridae lineages organization, which will be valuable in future updates to the taxonomy of the family since the current official classification does not capture all the Marseilleviridae known diversity.

Development and evaluation of an artificial intelligence for bacterial growth monitoring in clinical bacteriology.

In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.

Aberrant Bodies: An Alternative Metabolic Homeostasis Allowing Survivability?

The Chlamydiae phylum is comprised of obligate intracellular bacteria including human pathogens such as Chlamydia trachomatis and lesser-known Chlamydia-related bacteria like Waddlia chondrophila or Simkania negevensis. Despite broad differences, these bacteria share a similar development including a persistent state induced using stressors such as immune responses, nutrient starvation, or penicillin introduction. In microbiology, this persistent state is identified by enlarged bacteria, called aberrant bodies, which are unable to divide but are able to survive and resume the developmental cycle upon clearance of the stressor. Clinically, chlamydial persistence is thought to be linked to chronic disease and long-term infections with pathogenic strains. This review aims to share and discuss the latest discoveries made on the little-known mechanisms that take place during stress response. The results indicate that an inter-linked homeostasis between iron and tryptophan is required for effective bacterial proliferation. During stress, Chlamydiae attempt to compensate by inducing tight regulations of the tryptophan and iron acquisition operons. These compensations allow bacterial survival but result in the halting of cell division. As cell division is tightly linked to peptidoglycan synthesis and regulation, treatment with ß-lactamase inhibitors can also exhibit an aberrant body phenotype.

Accurate and rapid antibiotic susceptibility testing using a machine learning-assisted nanomotion technology platform.

Antimicrobial resistance (AMR) is a major public health threat, reducing treatment options for infected patients. AMR is promoted by a lack of access to rapid antibiotic susceptibility tests (ASTs). Accelerated ASTs can identify effective antibiotics for treatment in a timely and informed manner. We describe a rapid growth-independent phenotypic AST that uses a nanomotion technology platform to measure bacterial vibrations. Machine learning techniques are applied to analyze a large dataset encompassing 2762 individual nanomotion recordings from 1180 spiked positive blood culture samples covering 364 Escherichia coli and Klebsiella pneumoniae isolates exposed to cephalosporins and fluoroquinolones. The training performances of the different classification models achieve between 90.5 and 100% accuracy. Independent testing of the AST on 223 strains, including in clinical setting, correctly predict susceptibility and resistance with accuracies between 89.5% and 98.9%. The study shows the potential of this nanomotion platform for future bacterial phenotype delineation.

How much should we sequence? An analysis of the Swiss SARS-CoV-2 surveillance effort.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

Chlamydia pneumoniae Upsurge at Tertiary Hospital, Lausanne, Switzerland.

Chlamydia pneumoniae infection cases have usually accounted for <1.5% of community-acquired respiratory tract infections. Currently, Lausanne, Switzerland is experiencing a notable upsurge in cases, with 28 reported within a span of a few months. This upsurge in cases highlights the need for heightened awareness among clinicians.

Proposed Framework for Conducting Clinically Relevant Translational Biomarker Research for the Diagnosis, Prognosis and Management of Sepsis.

Although the diagnosis of sepsis requires the identification of the three components of infection, a systemic inflammation response, and organ dysfunction, there is currently no consensus on gold-standard criteria. There are however suggested tools and tests, which have been proposed in international guidelines, including those produced by the Surviving Sepsis Campaign. Biomarkers play an important role in these tools and tests, and numerous heterogeneous studies have been performed to evaluate their respective clinical utility. Our review of the current practice shows that no biomarkers of infection, systemic inflammation response, organ dysfunction and sepsis are currently specifically recommended, which is probably due to the lack of standardization of studies. We therefore propose to define a framework for conducting clinically relevant translational biomarker research and seek to establish ideal criteria that can be applied to an infection, systemic inflammation response, organ dysfunction and sepsis biomarkers, which can enable early screening of sepsis, diagnosis of sepsis at the time of clinical suspicion and monitoring of sepsis treatment efficacy.

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

Core genome multilocus sequence typing of Clostridioides difficile to investigate transmission in the hospital setting.

PURPOSE: Traditional epidemiological investigations of healthcare-associated Clostridioides difficile infection (HA-CDI) are often insufficient. This study aimed to evaluate a procedure that includes secondary isolation and genomic typing of single toxigenic colonies using core genome multilocus sequence typing (cgMLST) for the investigation of C. difficile transmission. METHODS: We analyzed retrospectively all toxigenic C. difficile-positive stool samples stored at the Lausanne University Hospital over 6 consecutive months. All isolates were initially typed and classified using a modified double-locus sequence typing (DLST) method. Genome comparison of isolates with the same DLST and clustering were subsequently performed using cgMLST. The electronic administrative records of patients with CDI were investigated for spatiotemporal epidemiological links supporting hospital transmission. A comparative descriptive analysis between genomic and epidemiological data was then performed. RESULTS: From January to June 2021, 86 C. difficile isolates were recovered from thawed samples of 71 patients. Thirteen different DLST types were shared by > 1 patient, and 13 were observed in single patients. A genomic cluster was defined as a set of isolates from different patients with ≤ 3 locus differences, determined by cgMLST. Seven genomic clusters were identified, among which plausible epidemiological links were identified in only 4/7 clusters. CONCLUSION: Among clusters determined by cgMLST analysis, roughly 40% included unexplained HA-CDI acquisitions, which may be explained by unidentified epidemiological links, asymptomatic colonization, and/or shared common community reservoirs. The use of DLST, followed by whole genome sequencing analysis, is a promising and cost-effective stepwise approach for the investigation of CDI transmission in the hospital setting.

Spontaneous Aberrant Bodies Formation in Human Pneumocytes Infected with Estrella lausannensis.

Estrella lausannensis, a Chlamydia-related bacterium isolated from a Spanish river, is considered as a possible emerging human pathogen. Indeed, it was recently demonstrated to multiply in human macrophages, resisting oxidative burst and causing a strong cytopathic effect. In addition, a preliminary study highlighted a correlation between antibody response to E. lausannensis and pneumonia in children. To clarify the pathogenic potential of these bacteria, we infected a human pneumocyte cell line with E. lausannensis and assessed its replication and cytopathic effect using quantitative real-time PCR and immunofluorescence, as well as confocal and electron microscopy. Our results demonstrated that E. lausannensis enters and replicates rapidly in human pneumocytes, and that it causes a prompt lysis of the host cells. Furthermore, we reported the spontaneous formation of aberrant bodies, a form associated with persistence in Chlamydiae, suggesting that E. lausannensis infection could cause chronic disorders in humans.

Ticks and Chlamydia-Related Bacteria in Swiss Zoological Gardens Compared to in Contiguous and Distant Control Areas.

Ticks are vectors of numerous agents of medical importance and may be infected by various Chlamydia-related bacteria, such as members of Parachlamydiaceae and Rhabdochlamydiaceae families, which are sharing the same biphasic life cycle with the pathogenic Chlamydia. However, the veterinary importance of ticks and of their internalized pathogens remains poorly studied. Thus, we wondered (i) whether the prevalence of ticks was higher in zoological gardens than in control areas with similar altitude, vegetation, humidity and temperature, and (ii) whether the presence of Chlamydia-related bacteria in ticks may vary according to the environment in which the ticks are collected. A total of 212 Ixodes ricinus ticks were collected, and all were tested for the presence of DNA from any member of the Chlamydiae phylum using a pan-Chlamydiae quantitative PCR (qPCR). We observed a higher prevalence of ticks outside animal enclosures in both zoos, compared to in enclosures. Tick prevalence was also higher outside zoos, compared to in enclosures. With 30% (3/10) of infected ticks, the zoological gardens presented a prevalence of infected ticks that was higher than that in contiguous areas (13.15%, 10/76), and higher than the control distant areas (8.65%, 9/104). In conclusion, zoological gardens in Switzerland appear to contain fewer ticks than areas outside zoological gardens. However, ticks from zoos more often contain Chlamydia-like organisms than ticks from contiguous or distant control areas.

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.

Temporal changes in fecal microbiota of patients infected with COVID-19: a longitudinal cohort.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a multifaceted disease potentially responsible for various clinical manifestations including gastro-intestinal symptoms. Several evidences suggest that the intestine is a critical site of immune cell development, gut microbiota could therefore play a key role in lung immune response. We designed a monocentric longitudinal observational study to describe the gut microbiota profile in COVID-19 patients and compare it to a pre-existing cohort of ventilated non-COVID-19 patients. METHODS: From March to December 2020, we included patients admitted for COVID-19 in medicine (43 not ventilated) or intensive care unit (ICU) (14 ventilated) with a positive SARS-CoV-2 RT-PCR assay in a respiratory tract sample. 16S metagenomics was performed on rectal swabs from these 57 COVID-19 patients, 35 with one and 22 with multiple stool collections. Nineteen non-COVID-19 ICU controls were also enrolled, among which 14 developed ventilator-associated pneumonia (pneumonia group) and five remained without infection (control group). SARS-CoV-2 viral loads in fecal samples were measured by qPCR. RESULTS: Although similar at inclusion, Shannon alpha diversity appeared significantly lower in COVID-19 and pneumonia groups than in the control group at day 7. Furthermore, the microbiota composition became distinct between COVID-19 and non-COVID-19 groups. The fecal microbiota of COVID-19 patients was characterized by increased Bacteroides and the pneumonia group by Prevotella. In a distance-based redundancy analysis, only COVID-19 presented significant effects on the microbiota composition. Moreover, patients in ICU harbored increased Campylobacter and decreased butyrate-producing bacteria, such as Lachnospiraceae, Roseburia and Faecalibacterium as compared to patients in medicine. Both the stay in ICU and patient were significant factors affecting the microbiota composition. SARS-CoV-2 viral loads were higher in ICU than in non-ICU patients. CONCLUSIONS: Overall, we identified distinct characteristics of the gut microbiota in COVID-19 patients compared to control groups. COVID-19 patients were primarily characterized by increased Bacteroides and decreased Prevotella. Moreover, disease severity showed a negative correlation with butyrate-producing bacteria. These features could offer valuable insights into potential targets for modulating the host response through the microbiota and contribute to a better understanding of the disease's pathophysiology. TRIAL REGISTRATION: CER-VD 2020-00755 (05.05.2020) & 2017-01820 (08.06.2018).

Use of gene sequences as type for naming prokaryotes: Recommendations of the international committee on the taxonomy of chlamydiae.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Corrigendum: A MALDI-TOF MS library for rapid identification of human commensal gut bacteria from the class Clostridia.

[This corrects the article DOI: 10.3389/fmicb.2023.1104707.].

Nanomotion technology in combination with machine learning: a new approach for a rapid antibiotic susceptibility test for Mycobacterium tuberculosis.

Nanomotion technology is a growth-independent approach that can be used to detect and record the vibrations of bacteria attached to cantilevers. We have developed a nanomotion-based antibiotic susceptibility test (AST) protocol for Mycobacterium tuberculosis (MTB). The protocol was used to predict strain phenotype towards isoniazid (INH) and rifampicin (RIF) using a leave-one-out cross-validation (LOOCV) and machine learning techniques. This MTB-nanomotion protocol takes 21 h, including cell suspension preparation, optimized bacterial attachment to functionalized cantilever, and nanomotion recording before and after antibiotic exposure. We applied this protocol to MTB isolates (n = 40) and were able to discriminate between susceptible and resistant strains for INH and RIF with a maximum sensitivity of 97.4% and 100%, respectively, and a maximum specificity of 100% for both antibiotics when considering each nanomotion recording to be a distinct experiment. Grouping recordings as triplicates based on source isolate improved sensitivity and specificity to 100% for both antibiotics. Nanomotion technology can potentially reduce time-to-result significantly compared to the days and weeks currently needed for current phenotypic ASTs for MTB. It can further be extended to other anti-TB drugs to help guide more effective TB treatment.