Extensive Comparative Genomic Analysis of Enterococcus faecalis and Enterococcus faecium Reveals a Direct Association between the Absence of CRISPR-Cas Systems, the Presence of Anti-Endonuclease (ardA) and the Acquisition of Vancomycin Resistance in E. faecium.

Here, we performed a comparative genomic analysis of all available genomes of E. faecalis (n = 1591) and E. faecium (n = 1981) and investigated the association between the presence or absence of CRISPR-Cas systems, endonuclease/anti-endonuclease systems and the acquisition of antimicrobial resistance, especially vancomycin resistance genes. Most of the analysed Enterococci were isolated from humans and less than 14% of them were from foods and animals. We analysed and detected CRISPR-Cas systems in 75.36% of E. faecalis genomes and only 4.89% of E. faecium genomes with a significant difference (p-value < 10-5). We found a negative correlation between the number of CRISPR-Cas systems and genome size (r = -0.397, p-value < 10-5) and a positive correlation between the genome %GC content and the number of CRISPR-Cas systems (r = 0.215, p-value < 10-5). Our findings showed that the presence of the anti-endonuclease ardA gene may explain the decrease in the number of CRISPR-Cas systems in E. faecium, known to deactivate the endonucleases' protective activities and enable the E. faecium genome to be versatile in acquiring mobile genetic elements, including carriers of antimicrobial resistance genes, especially vanB. Most importantly, we observed that there was a direct association between the absence of CRISPR-Cas, the presence of the anti-CRISPR ardA gene and the acquisition of vancomycin resistance genes.

HCK and ABAA: A Newly Designed Pipeline to Improve Fungi Metabarcoding Analysis.

INTRODUCTION: The fungi ITS sequence length dissimilarity, non-specific amplicons, including chimaera formed during Polymerase Chain Reaction (PCR), added to sequencing errors, create bias during similarity clustering and abundance estimation in the downstream analysis. To overcome these challenges, we present a novel approach, Hierarchical Clustering with Kraken (HCK), to classify ITS1 amplicons and Abundance-Base Alternative Approach (ABAA) pipeline to detect and filter non-specific amplicons in fungi metabarcoding sequencing datasets. MATERIALS AND METHODS: We compared the performances of both pipelines against QIIME, KRAKEN, and DADA2 using publicly available fungi ITS mock community datasets and using BLASTn as a reference. We calculated the Precision, Recall, F-score using the True-Positive, False-positive, and False-negative estimation. Alpha diversity (Chao1 and Shannon metrics) was also used to evaluate the diversity estimation of our method. RESULTS: The analysis shows that ABAA reduced the number of false-positive with all metabarcoding methods tested, and HCK increases precision and recall. HCK, coupled with ABAA, improves the F-score and bring alpha diversity metric value close to that of the BLASTn alpha diversity values when compared to QIIME, KRAKEN, and DADA2. CONCLUSION: The developed HCK-ABAA approach allows better identification of the fungi community structures while avoiding use of a reference database for non-specific amplicons filtration. It results in a more robust and stable methodology over time. The software can be downloaded on the following link: https://bitbucket.org/GottySG36/hck/src/master/.

Successful treatment and digestive decolonisation of a patient with osteitis caused by a carbapenemase-producing Klebsiella pneumoniae isolate harbouring both NDM-1 and OXA-48 enzymes.

OBJECTIVES: Carbapenem resistance in Klebsiella pneumoniae is an increasing problem worldwide and infections caused by this bacterium can be difficult to treat. This study reported the case of a patient from Romania, who was hospitalised in Bulgaria after an accident trauma. He then came to France for treatment of an osteitis caused by a Klebsiella pneumoniae carrying both blaNDM-1 and blaOXA-48. METHOD: The resistome of this extremely drug-resistant bacterium was analysed both with phenotypic (large antibiotic susceptibility testing) and genomic methods (genome sequencing). The genetic environment of the two carbapenemases was studied. RESULTS: Klebsiella pneumoniae ST307 carrying both a blaNDM-1 and blaOXA-48 gene was located on two different plasmids: Inc L/M and IncFII. The patient was successfully treated by a combination of intravenous colistin (9 MUI, then 4.5 MUI bd), intravenous fosfomycin (4g tds) and oral doxycycline (100mg bd) for 3 months. Faecal microbiota transplantation was successfully conducted for stool carriage. CONCLUSION: The ST307 type is becoming endemic in hospital environments and is frequently associated with carbapenem resistance. Treatment of infection caused by multidrug-resistant bacteria is a clinical challenge, and the use of old antibiotics associated with screening and decolonisation of the reservoirs can be an efficient therapeutic alternative.

A tuberculosis nationwide prevalence survey in Gambia, 2012.

OBJECTIVE: To estimate the population prevalence of active pulmonary tuberculosis in Gambia. METHODS: Between December 2011 and January 2013, people aged ≥ 15 years participating in a nationwide, multistage cluster survey were screened for active pulmonary tuberculosis with chest radiography and for tuberculosis symptoms. For diagnostic confirmation, sputum samples were collected from those whose screening were positive and subjected to fluorescence microscopy and liquid tuberculosis cultures. Multiple imputation and inverse probability weighting were used to estimate tuberculosis prevalence. FINDINGS: Of 100 678 people enumerated, 55 832 were eligible to participate and 43 100 (77.2%) of those participated. A majority of participants (42 942; 99.6%) were successfully screened for symptoms and by chest X-ray. Only 5948 (13.8%) were eligible for sputum examination, yielding 43 bacteriologically confirmed, 28 definite smear-positive and six probable smear-positive tuberculosis cases. Chest X-ray identified more tuberculosis cases (58/69) than did symptoms alone (43/71). The estimated prevalence of smear-positive and bacteriologically confirmed pulmonary tuberculosis were 90 (95% confidence interval, CI: 53-127) and 212 (95% CI: 152-272) per 100 000 population, respectively. Tuberculosis prevalence was higher in males (333; 95% CI: 233-433) and in the 35-54 year age group (355; 95% CI: 219-490). CONCLUSION: The burden of tuberculosis remains high in Gambia but lower than earlier estimates of 490 per 100 000 population in 2010. Less than half of all cases would have been identified based on smear microscopy results alone. Successful control efforts will require interventions targeting men, increased access to radiography and more accurate, rapid diagnostic tests.

Face mask sampling for the detection of Mycobacterium tuberculosis in expelled aerosols.

BACKGROUND: Although tuberculosis is transmitted by the airborne route, direct information on the natural output of bacilli into air by source cases is very limited. We sought to address this through sampling of expelled aerosols in face masks that were subsequently analyzed for mycobacterial contamination. METHODS: In series 1, 17 smear microscopy positive patients wore standard surgical face masks once or twice for periods between 10 minutes and 5 hours; mycobacterial contamination was detected using a bacteriophage assay. In series 2, 19 patients with suspected tuberculosis were studied in Leicester UK and 10 patients with at least one positive smear were studied in The Gambia. These subjects wore one FFP30 mask modified to contain a gelatin filter for one hour; this was subsequently analyzed by the Xpert MTB/RIF system. RESULTS: In series 1, the bacteriophage assay detected live mycobacteria in 11/17 patients with wearing times between 10 and 120 minutes. Variation was seen in mask positivity and the level of contamination detected in multiple samples from the same patient. Two patients had non-tuberculous mycobacterial infections. In series 2, 13/20 patients with pulmonary tuberculosis produced positive masks and 0/9 patients with extrapulmonary or non-tuberculous diagnoses were mask positive. Overall, 65% of patients with confirmed pulmonary mycobacterial infection gave positive masks and this included 3/6 patients who received diagnostic bronchoalveolar lavages. CONCLUSION: Mask sampling provides a simple means of assessing mycobacterial output in non-sputum expectorant. The approach shows potential for application to the study of airborne transmission and to diagnosis.