Towards the reconstruction of a global TB history using a new pipeline "TB-Annotator".

Mycobacterium tuberculosis complex (MTBC) has a population structure consisting of 9 human and animal lineages. The genomic diversity within these lineages is a pathogenesis factor that affects virulence, transmissibility, host response, and antibiotic resistance. Hence it is important to develop improved information systems for tracking and understanding the spreading and evolution of genomes. We present results obtained thanks to a new informatics platform for computational biology of MTBC, that uses a convenience sample from public/private SRAs, designated as TB-Annotator. Version 1 was a first interactive graphic-based web tool based on 15,901 representative genomes. Version 2, still interactive, is a more sophisticated database, developed using the Snakemake Workflow Management System (WMS) that allows an unsupervised global and scalable analysis of the content of the USA National Center for Biotechnology Information Short Read Archives database. This platform analyzes nucleotide variants, the presence/absence of genes, known regions of difference and detect new deletions, the insertion sites of mobile genetic elements, and allows phylogenetic trees to be built, imported in a graphical interface and interactively analyzed between the data and the tree. The objective of TB-Annotator is triple: detect recent epidemiological links, reconstruct distant phylogeographical histories as well as perform more complex phenotypic/genotypic Genome-Wide Association Studies (GWAS). In this paper, we compare the various taxonomic SNPs-based labels and hierarchies previously described in recent reference papers for L1, and present a comparative analysis that allows identification of alias and thus provides the basis of a future unifying naming scheme for L1 sublineages. We present a global phylogenetic tree built with RAxML-NG, and one on L2; at the time of writing, we characterized about 200 sublineages, with many new ones; a detail tree for Modern L2 and a hierarchical scheme allowing to facilitate L2 lineage assignment are also presented.

Performances of bioinformatics tools for the analysis of sequencing data of Mycobacterium tuberculosis complex strains.

Whole-genome sequencing of Mycobacterium tuberculosis complex (MTBC) strains is a rapidly growing tool to obtain results regarding the resistance and phylogeny of the strains. We evaluated the performances of two bioinformatics tools for the analysis of whole-genome sequences of MTBC strains. Two hundred and twenty-seven MTBC strains were isolated and whole-genome sequenced at the laboratory of Avicenne Hospital between 2015 and 2021. We investigated the resistance and susceptibility status of strains using two online tools, Mykrobe and PhyResSE. We compared the genotypic and phenotypic resistance results obtained by drug susceptibility testing. Unlike with the Mykrobe tool, sequencing quality data were obtained using PhyResSE: average coverage of 98% and average depth of 119X. We found a similar concordance between phenotypic and genotypic results when determining susceptibility to first-line anti-tuberculosis drugs (95%) with both tools. The sensitivity and specificity of each tool compared to the phenotypic method were respectively 72% [52-87] and 98% [96-99] for Mykrobe and 76% [57-90] and 97% [94-99] for PhyResSE. Mykrobe and PhyResSE were easy to use and efficient. These platforms are accessible to people not trained in bioinformatics and constitute a complementary approach to phenotypic methods for the study of MTBC strains.

Use of Whole-Genome Sequencing to Explore Mycobacterium tuberculosis Complex Circulating in a Hotspot Department in France.

The Seine-Saint-Denis is the French metropolitan department with the highest incidence of tuberculosis (TB). Our aim was to explore epidemiological and phylogenetic characteristics of TB strains in this hotspot department. We performed WGS on 227 strains of Mycobacterium tuberculosis complex isolated from patients at the Avicenne Hospital from 2016 to 2021 and randomly selected to represent the clinical diversity of French TB localization. Clinical and demographic data were recorded for each TB patient. The mean age of patients was 36 years old. They came from Africa (44%), Asia (27%), Europe (26%) and America (3%). Strains isolated from extrapulmonary samples were associated with Asian patients, whereas strains isolated from pulmonary samples were associated with European patients. We observed a high level of lineage diversity in line with the known worldwide diversity. Interestingly, lineage 3 was associated with lymph node TB. Additionally, the sensitivity of WGS for predicting resistance was 100% for rifampicin, isoniazid and ethambutol and 66.7% for pyrazinamide. The global concordance with drug-susceptibility testing using the phenotypic approach was 97%. In microbiology laboratories, WGS turns out to be an essential tool for better understanding local TB epidemiology, with direct access to circulating lineage identification and to drug susceptibilities to first- and second-line anti-TB drugs.

Prevalence and risk factors for carriage of extended-spectrum ß-lactamase-producing enterobacteriaceae in rehabilitation wards in France.

OBJECTIVES: Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) are a major public health concern worldwide. Little is known about the prevalence of ESBL-PE colonization in rehabilitation wards in France. Our aim was to determine the prevalence of ESBL-PE colonization in rehabilitation wards in the Parisian area and to identify potential risk factors for ESBL-PE carriage. PATIENTS AND METHODS: This one-day prospective study was performed in three rehabilitation wards in Paris, France, between September 1, 2016, and June 26, 2017. Rectal samples were collected for microbial analysis from patients who were present at 8am and all isolates recovered were identified by mass spectrometry. The presence of ESBL-PE was confirmed using a double-disk synergy test according to EUCAST recommendations. Risk factors for colonization were determined by univariate and multivariate analyses. RESULTS: A total of 136 patients were analyzed (50.7% of female patients; median age 71 years). Twenty-eight patients (20.6%) were colonized with ESBL-PE on the day of sampling. Escherichia coli was identified in 15 (50%) cases and Klebsiella pneumoniae in six (20%). None of the variables studied was significantly associated with a higher risk of ESBL-PE colonization. However, there was a tendency for a higher risk of ESBL-PE colonization with dementia (OR = 6.116 [95%CI: 0.92-40.57]; p = 0.06) and diabetes with complications (OR = 2.853 [95%CI: 1-8.16]; p = 0.05). CONCLUSIONS: Patients in rehabilitation wards showed a high rate of ESBL-PE colonization (21%). Continuous monitoring of antibiotic resistance among potentially pathogenic bacteria, including ESBL-PE, is crucial as colonized patients represent an important reservoir for transmission after discharge outside the hospital setting.

Molecular detection of isoniazid monoresistance improves tuberculosis treatment: A retrospective cohort in France.

OBJECTIVES: Isoniazid-monoresistant tuberculosis (HR-TB) requires early diagnosis and adapted treatment to achieve optimal outcomes. The primary aim of the study was to assess the impact of the implementation of rapid diagnostic tests on HR-TB treatment in France. METHODS: We designed a retrospective multicentre study including consecutive HR-TB patients diagnosed in 2016 and 2017. Implementation of a molecular assay detecting isoniazid resistance directly on a clinical sample was recorded. The association between early implementation of such assays and adequate treatment was assessed by a multivariable Cox proportional hazards model. RESULTS: Overall, 99 HR-TB patients were included from 20 University Hospitals. Among all smear-positive HR-TB patients, only 26% beneficiated from early molecular HR detection. This detection was independently associated with shorter time to adequate treatment (HR = 2.0 [1.1-3.8], p = 0.03). CONCLUSION: In our study, molecular detection of HR on an initial sample was independently associated with earlier treatment adaptation.

Isoniazid-monoresistant tuberculosis in France: Risk factors, treatment outcomes and adverse events.

OBJECTIVES: Isoniazid-monoresistant tuberculosis (HR-TB) is the most prevalent form of drug-resistant TB worldwide and in France and is associated with poorer treatment outcomes compared with drug-susceptible TB (DS-TB). The objective of this study was to determine the characteristics of HR-TB patients in France and to compare outcomes and safety of treatment for HR-TB and DS-TB. METHODS: We performed a case-control multicenter study to identify risk factors associated with HR-TB and compare treatment outcomes and safety between HR-TB patients and DS-TB patients. RESULTS: Characteristics of 99 HR-TB patients diagnosed and treated in the university hospitals of Paris, Lille, Caen and Strasbourg were compared with 99 DS-TB patients. Female sex (OR = 2.2; 1.0-4.7), birth in the West-Pacific World Health Organization region (OR = 4.6; 1.1-18.7) and resistance to streptomycin (OR = 77.5; 10.1-594.4) were found to be independently associated with HR-TB. Rates of treatment success did not differ significantly between HR-TB and DS-TB. CONCLUSIONS: Factors associated with HR-TB are not significant enough to efficiently screen TB patients at risk of HR-TB. The systematic implementation of rapid molecular testing on clinical samples remains the only effective way to make the early diagnosis of HR-TB and adapt treatment.

Temocillin susceptibility among Enterobacterales strains recovered from blood culture in France.

Temocillin is used for several years in some European countries but, only since 2015 in France. We assessed the susceptibility of Enterobacterales strains isolated from blood culture 1 year before (2014) and 2 years after (2017) its use in France. 1,387 strains were included by 17 clinical laboratories located throughout France: 363 in 2014 and 1,024 in 2017. The rate of resistance to temocillin was 4.6% and 26.7% in 3rd generation cephalosporin (3GC) susceptible and resistant strains respectively. Cephalosporinase-overproducer (COPE) strains were significantly more resistant to temocillin (37.7%) than ESBL-producer (ESBL-PE) (23.5%) (P < 0.01). The rate of temocillin resistance was correlated to the number of inactive beta-lactams. The rate of resistance to temocillin trend to increase from 13.9% in 2014 to 23.9% in 2017 (P < 0.01). Temocillin remains highly active against Enterobacterales but the trend in resistance should be assessed over time.

A recurrent lung abscess caused by delayed diagnosis of unique co-infection with Abiotrophia defectiva.

We report the case of a lung abscess due to Prevotella baroniae with a co-infection by Abiotrophia defective, which is a 'nutritionally variant streptococci' (NVS), in a 48-year-old patient. The delayed diagnosis of this co-infection led to multiple failures of medical treatment and need for surgery. Pathogenicity of these bacteria is well known, particularly in endocarditis, but not in lung infection. In pulmonary abscesses, co-infection with NVS is difficult to detect. It may explain some medical treatment failures. This case highlights the importance to systematically search for and consider NVS in such clinical contexts.

Molecular identification of Actinomadura madurae isolated from a patient originally from Algeria; observations from a case report.

BACKGROUND: Mycetoma is a chronic granulomatous subcutaneous infection caused by anaerobic pseudofilamentous bacteria or fungi. It is commonly prevalent in tropical and subtropical countries. Men are more susceptible to the disease due to greater participation in agricultural works. Mycetoma commonly involves lower extremities, wherein untreated cases lead to aggressive therapeutic choices, such as amputation of the affected body organs and consequently lifelong disability. CASE PRESENTATION: In this report, we present the rare case of a 58-year-old man, originally from Algeria with a left foot chronic tumefaction of 5 years. In the initial clinical examination, mycetoma was diagnosed based on tumefaction and the presence of multiple sinuses with the emission of white grains. The latter was observed via direct examination. The histopathological analysis demonstrated an actinomycetoma caused by bacteria, as the etiological agent. Imaging showed a bone involvement with osteolysis at the levels of 2nd to 4th metatarsal diaphysis. The mycological and bacterial cultures were both negative. For an accurate diagnosis, the obtained grains were subjected to molecular analysis, targeting the 16S-rDNA gene. Molecular identification yielded Actinomadura madurae as the causal agent, and 800/160 mg of trimethoprim/sulfamethoxazole was prescribed twice a day for 1 year, as a treatment. CONCLUSION: Considering low information about this disease, especially in non-endemic areas, it is of high importance to enhance the knowledge and awareness of clinicians and healthcare providers, in particular in the countries with immigration issues.

Fatal Legionella pneumophila serogroup 1 pleural empyema: A case report.

BACKGROUND: Legionella pneumophila (L. pneumophila) is a gram-negative intracellular bacillus composed of sixteen different serogroups. It is mostly known to cause pneumonia in individuals with known risk factors as immunocompromised status, tobacco use, chronic organ failure or age older than 50 years. Although parapneumonic pleural effusion is frequent in legionellosis, pleural empyema is very uncommon. In this study, we report a case of fatal pleural empyema caused by L. pneumophila serogroup 1 in an 81-year-old man with multiple risk factors. CASE SUMMARY: An 81-year-old man presented to the emergency with a 3 wk dyspnea, fever and left chest pain. His previous medical conditions were chronic lymphocytic leukemia, diabetes mellitus, chronic kidney failure, hypertension and hyperlipidemia, without tobacco use. Chest X-ray and comouted tomography-scan confirmed a large left pleural effusion, which puncture showed a citrine exudate with negative standard bacterial cultures. Despite intravenous cefotaxime antibiotherapy, patient's worsening condition after 10 d led to thoracocentesis and evacuation of 2 liters of pus. The patient progressively developed severe hypoxemia and multiorgan failure occurred. The patient was treated by antibiotherapy with cefepime and amikacin and with adequate symptomatic shock treatment, but died of uncontrolled sepsis. The next day, cultures of the surgical pleural liquid samples yielded L. pneumophila serogroup 1, consistent with the diagnosis of pleural legionellosis. CONCLUSION: L. pneumophila should be considered in patients with multiple risk factors and undiagnosed pleural empyema unresponsive to conventional antibiotherapy.

Proposition of a safe Mycobacterium tuberculosis complex denaturation method that does not compromise the integrity of DNA for whole-genome sequencing.

Whole-genome sequencing plays now a leading role in epidemiologic studies of tuberculosis. DNA extraction of Mycobacterium tuberculosis complex (MTBC) requires complete inactivation of the strains, to be handled for further molecular procedures. In this study we compared two chloroform-based denaturation methods (one with a step of heat killing, one without) to a traditional heat inactivation method. Our results showed that 40% of the strains of MTBC treated by the traditional protocol resulted in a positive culture whereas no culture was observed with the two chloroform-based protocols. The DNA extracts obtained with chloroform-based protocols preparation were successfully used for whole-genome sequencing. We recommend inactivation with our rapid and efficient denaturation method using chloroform without heat killing which met our expectations and biosecurity requirements.

Using SNOMED-CT to Help the Transition from Microbiological Data to ICD-10 Sepsis Codes.

Assigning ICD-10 code of sepsis in regard of a pathogenic bacterium found in an haemoculture requires knowledge of microbiology because of the difference of granularity. The aim of this paper is to automate this coding thanks to the use of SNOMED-CT. A dichotomous classification of bacteria causing sepsis has been generated in respect of ICD-10. Our algorithm follows this and explores SNOMED-CT to assign the right ICD-10 code of the sepsis. Applied to a list of 164 bacteria, the system has an error rate of 1.22 %.

Specialization of small non-conjugative plasmids in Escherichia coli according to their family types.

We undertook a comprehensive comparative analysis of a collection of 30 small (<25 kb) non-conjugative Escherichia coli plasmids previously classified by the gene sharing approach into 10 families, as well as plasmids found in the National Center for Biotechnology Information (NCBI) nucleotide database sharing similar genomic sequences. In total, 302 mobilizable (belonging to 2 MOBrep and 5 MOBRNA families) and 106 non-transferable/relaxase-negative (belonging to three ReLRNA families) plasmids were explored. The most striking feature was the specialization of the plasmid family types that was not related to their transmission mode and replication system. We observed a range of host strain specificity, from narrow E. coli host specificity to broad host range specificity, including a wide spectrum of Enterobacteriaceae. We found a wide variety of toxin/antitoxin systems and colicin operons in the plasmids, whose numbers and types varied according to the plasmid family type. The plasmids carried genes conferring resistance spanning almost all of the antibiotic classes, from those to which resistance developed early, such as sulphonamides, to those for which resistance has only developed recently, such as colistin. However, the prevalence of the resistance genes varied greatly according to the family type, ranging from 0 to 100 %. The evolutionary history of the plasmids based on the family type core genes showed variability within family nucleotide divergences in the range of E. coli chromosomal housekeeping genes, indicating long-term co-evolution between plasmids and host strains. In rare cases, a low evolutionary divergence suggested the massive spread of an epidemic plasmid. Overall, the importance of these small non-conjugative plasmids in bacterial adaptation varied greatly according to the type of family they belonged to, with each plasmid family having specific hosts and genetic traits.

The Arginine Deiminase Operon Is Responsible for a Fitness Trade-Off in Extended-Spectrum-ß-Lactamase-Producing Strains of Escherichia coli.

We previously identified an operon involved in an arginine deiminase (ADI) pathway (arc operon) on a CTX-M-producing plasmid from an O102-ST405 strain of Escherichia coli As the ADI pathway was shown to be involved in the virulence of various Gram-positive bacteria, we tested whether the ADI pathway could be involved in the epidemiological success of extended-spectrum-ß-lactamase (ESBL)-producing E. coli strains. We studied two collections of human E. coli isolated in France (n = 493) and England (n = 1,509) and show that the prevalence of the arc operon (i) is higher in ESBL-producing strains (12.1%) than in nonproducers (2.5%), (ii) is higher in CTX-M-producing strains (16%) than in other ESBL producers (3.5%), and (iii) increased over time in ESBL-producing strains from 0% before 2000 to 43.3% in 2011 to 2012. The arc operon, found in strains from various phylogenetic backgrounds, is carried by IncF plasmids (85%) or chromosomes (15%) in regions framed by numerous insertion sequences, indicating multiple arrivals. Competition experiments showed that the arc operon enhances fitness of the strain in vitro in lysogeny broth with arginine. In vivo competition experiments showed that the arc operon is advantageous for the strain in a mouse model of urinary tract infection (UTI), whereas it is a burden in a mouse model of intestinal colonization. In summary, we have identified a trait linked to CTX-M-producing strains that is responsible for a trade-off between two main E. coli lifestyles, UTI and gut commensalism. This trait alone cannot explain the wide spread of ESBLs in E. coli but merits epidemiological surveillance.

Pneumonia-Specific Escherichia coli with Distinct Phylogenetic and Virulence Profiles, France, 2012-2014.

In a prospective, nationwide study in France of Escherichia coli responsible for pneumonia in patients receiving mechanical ventilation, we determined E. coli antimicrobial susceptibility, phylotype, O-type, and virulence factor gene content. We compared 260 isolates with those of 2 published collections containing commensal and bacteremia isolates. The preponderant phylogenetic group was B2 (59.6%), and the predominant sequence type complex (STc) was STc73. STc127 and STc141 were overrepresented and STc95 underrepresented in pneumonia isolates compared with bacteremia isolates. Pneumonia isolates carried higher proportions of virulence genes sfa/foc, papGIII, hlyC, cnf1, and iroN compared with bacteremia isolates. Virulence factor gene content and antimicrobial drug resistance were higher in pneumonia than in commensal isolates. Genomic and phylogenetic characteristics of E. coli pneumonia isolates from critically ill patients indicate that they belong to the extraintestinal pathogenic E. coli pathovar but have distinguishable lung-specific traits.

Oropharyngeal Bacterial Colonization after Chlorhexidine Mouthwash in Mechanically Ventilated Critically Ill Patients.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Oropharyngeal care with chlorhexidine to prevent ventilator-associated pneumonia is currently questioned, and exhaustive microbiologic data assessing its efficacy are lacking. The authors therefore aimed to study the effect of chlorhexidine mouthwash on oropharyngeal bacterial growth, to determine chlorhexidine susceptibility of these bacteria, and to measure chlorhexidine salivary concentration after an oropharyngeal care. METHODS: This observational, prospective, single-center study enrolled 30 critically ill patients under mechanical ventilation for over 48 h. Oropharyngeal contamination was assessed by swabbing the gingivobuccal sulcus immediately before applying 0.12% chlorhexidine with soaked swabs, and subsequently at 15, 60, 120, 240, and 360 min after. Bacterial growth and identification were performed, and chlorhexidine minimal inhibitory concentration of recovered pathogens was determined. Saliva was collected in 10 patients, at every timepoint, with an additional timepoint after 30 min, to measure chlorhexidine concentration. RESULTS: Two hundred fifty bacterial samples were analyzed and identified 48 pathogens including Streptococci (27.1%) and Enterobacteriaceae (20.8%). Oropharyngeal contamination before chlorhexidine mouthwash ranged from 10 to 10 colony-forming units (CFU)/ml in the 30 patients (median contamination level: 2.5·10 CFU/ml), and remained between 8·10 (lowest) and 3·10 CFU/ml (highest count) after chlorhexidine exposure. These bacterial counts did not decrease overtime after chlorhexidine mouthwash (each minute increase in time resulted in a multiplication of bacterial count by a coefficient of 1.001, P = 0.83). Viridans group streptococci isolates had the lowest chlorhexidine minimal inhibitory concentration (4 [4 to 8] mg/l); Enterobacteriaceae isolates had the highest ones (32 [16 to 32] mg/l). Chlorhexidine salivary concentration rapidly decreased, reaching 7.6 [1.8 to 31] mg/l as early as 60 min after mouthwash. CONCLUSIONS: Chlorhexidine oropharyngeal care does not seem to reduce bacterial oropharyngeal colonization in critically ill ventilated patients. Variable chlorhexidine minimal inhibitory concentrations along with low chlorhexidine salivary concentrations after mouthwash could explain this ineffectiveness, and thus question the use of chlorhexidine for ventilator-associated pneumonia prevention.

Extended-spectrum ß-lactamase-encoding genes are spreading on a wide range of Escherichia coli plasmids existing prior to the use of third-generation cephalosporins.

To understand the evolutionary dynamics of extended-spectrum ß-lactamase (ESBL)-encoding genes in Escherichia coli, we undertook a comparative genomic analysis of 116 whole plasmid sequences of human or animal origin isolated over a period spanning before and after the use of third-generation cephalosporins (3GCs) using a gene-sharing network approach. The plasmids included 82 conjugative, 22 mobilizable and 9 non-transferable plasmids and 3 P-like bacteriophages. ESBL-encoding genes were found on 64 conjugative, 6 mobilizable, 2 non-transferable plasmids and 2 P1-like bacteriophages, indicating that these last three types of mobile elements also play a role, albeit modest, in the diffusion of the ESBLs. The network analysis showed that the plasmids clustered according to their genome backbone type, but not by origin or period of isolation or by antibiotic-resistance type, including type of ESBL-encoding gene. There was no association between the type of plasmid and the phylogenetic history of the parental strains. Finer scale analysis of the more abundant clusters IncF and IncI1 showed that ESBL-encoding plasmids and plasmids isolated before the use of 3GCs had the same diversity and phylogenetic history, and that acquisition of ESBL-encoding genes had occurred during multiple independent events. Moreover, the blaCTX-M-15 gene, unlike other CTX-M genes, was inserted at a hot spot in a blaTEM-1-Tn2 transposon. These findings showed that ESBL-encoding genes have arrived on wide range of pre-existing plasmids and that the successful spread of blaCTX-M-15 seems to be favoured by the presence of well-adapted IncF plasmids that carry a Tn2-blaTEM-1 transposon.

Hypervirulent Klebsiella pneumoniae, a 5-year study in a French ICU.

PURPOSE: Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a leading cause of severe community-acquired pneumonia, liver abscess and disseminated infection in the Far East. Data regarding the incidence, clinical features and microbiological characteristics related to hvKp infections in the Western world are scarce. METHODOLOGY: The incidence, clinical features and microbiological characteristics of hvKp infections were investigated through a 5-year survey conducted in a single French intensive care unit. K. pneumoniae strains were screened for hypermucoviscosity based on a string test. Multilocus sequence typing and multiplex PCR analysis targeting virulence genes were performed on string test-positive strains. RESULTS: Over a 53-month period, a total of 59 infections due to K. pneumoniae were identified including 26 community-onset infections. Twelve hvKp infections were documented, accounting for 46.1 % of community-acquired K. pneumoniae. Community-acquired pneumonia (n=6), aspiration pneumonia (n=4) and liver abscess (n=2) represented initial sites and mode of infection. Compared to non-hvKp infections, patients with hvKp infections displayed higher rates of multi-organ failure (83.3 % vs 35.7 %; P=0.04), but mortality rates were not different (50 % vs 35 %; P=0.71). Strains K1/ST23 (n=5) and K2/ST86 (n=5) predominated. All hvKp strains displayed wild-type susceptibility. CONCLUSION: hvKp represent a potentially underestimated cause of fatal infections in the Western world.

Diagnosis in France of a Non-Toxigenic tox Gene-Bearing Strain of Corynebacterium diphtheriae in a Young Male Back From Senegal.

Cutaneous diphtheria is uncommon in Europe. In this study, we report a case of imported cutaneous infection due to a non-toxigenic but tox gene-bearing (NTTB) strain of Corynebacterium diphtheriae. The NTTB strains are recognized as emerging pathogens across Europe, and physicians and bacteriologists should be aware of the circulation of these strains.