Emergence of two novel sequence types (3366 and 3367) NDM-1- and OXA-48-co-producing K. pneumoniae in Italy.

The aim of this study was to analyze the alarming spread of NDM-1- and OXA-48-co-producing Klebsiella pneumoniae clinical isolates, collected between October 2016 and January 2018 in a neonatal intensive care unit of the University Hospital, Catania, Italy, through whole genome sequencing. All confirmed carbapenem-resistant K. pneumoniae (CRKp) isolates were characterized pheno- and geno-typically, as well as by whole genome sequencing (WGS). A total of 13 CRKp isolates were identified from 13 patients. Pulsed-field gel electrophoresis (PFGE) was performed, and the multilocus sequence typing (MLST) scheme used was based on the gene sequence as published on the MLST Pasteur website. Core genome MLST (cgMLST) was also performed. All isolates co-carried blaoxa-48 and blaNDM-1 genes located on different plasmids belonging to the IncM/L and IncA/C2 groups, respectively. The 13 strains had identical PFGE profiles. MLST and cgMLST showed that K. pneumoniae was dominated by CRKp ST101 and two novel STs (ST3666 and ST3367), identified after submission to the MLST database for ST assignment. All isolates shared the same virulence factors such as type 3 fimbriae, genes for yersiniabactin biosynthesis, yersiniabactin receptor, and iron ABC transporter. They carried the wzi137 variant associated with the K17 serotype. To the best of our knowledge, this is the first report of two novel STs, 3366 and 3367, NDM-OXA-48-co-producing K. pneumoniae clinical isolates, in Italy.

Automated detection of bacterial growth on 96-well plates for high-throughput drug susceptibility testing of Mycobacterium tuberculosis.

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the MICs to be elucidated. The three participating laboratories each inoculated 38 96-well plates with 15 known M. tuberculosis strains (including the standard H37Rv reference strain) and, after 2 weeks' incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. The AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (>30000) of samples of M. tuberculosis from patients over the next few years.

Updating the WHO target product profile for next-generation Mycobacterium tuberculosis drug susceptibility testing at peripheral centres.

There were approximately 10 million tuberculosis (TB) cases in 2020, of which 500,000 were drug-resistant. Only one third of drug-resistant TB cases were diagnosed and enrolled on appropriate treatment, an issue partly driven by a lack of rapid, accurate drug-susceptibility testing (DST) tools deployable in peripheral settings. In 2014, World Health Organization (WHO) published target product profiles (TPPs) which detailed minimal and optimal criteria to address high-priority TB diagnostic needs, including DST. Since then, the TB community's needs have evolved; new treatment regimens, changes in TB definitions, further emergence of drug resistance, technological advances, and changing end-users requirements have necessitated an update. The DST TPP's revision was therefore undertaken by WHO with the Stop TB Partnership New Diagnostics Working Group. We describe the process of updating the TPP for next-generation TB DST for use at peripheral centres, highlight key updates, and discuss guidance regarding technical and operational specifications.

A smooth tubercle bacillus from Ethiopia phylogenetically close to the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) includes several human- and animal-adapted pathogens. It is thought to have originated in East Africa from a recombinogenic Mycobacterium canettii-like ancestral pool. Here, we describe the discovery of a clinical tuberculosis strain isolated in Ethiopia that shares archetypal phenotypic and genomic features of M. canettii strains, but represents a phylogenetic branch much closer to the MTBC clade than to the M. canettii strains. Analysis of genomic traces of horizontal gene transfer in this isolate and previously identified M. canettii strains indicates a persistent albeit decreased recombinogenic lifestyle near the emergence of the MTBC. Our findings support that the MTBC emergence from its putative free-living M. canettii-like progenitor is evolutionarily very recent, and suggest the existence of a continuum of further extant derivatives from ancestral stages, close to the root of the MTBC, along the Great Rift Valley.