Implementation of the WHO Tricycle protocol for surveillance of extended-spectrum ß-lactamase producing Escherichia coli in humans, chickens, and the environment in Madagascar: a prospective genomic epidemiology study.

BACKGROUND: Antimicrobial resistance (AMR) is a major public health threat, affecting not only people but also animals and the environment. The One Health dimension of AMR is well known; however, data are lacking on the circulation of resistance-conferring genes, particularly in low-income countries. In 2017, WHO proposed a protocol called Tricycle, focusing on extended-spectrum ß-lactamase (ESBL)-Escherichia coli surveillance in the three sectors (humans, animals, and the environment). We implemented Tricycle in Madagascar to assess ESBL-E coli prevalence and describe intrasector and intersector circulation of ESBL-E coli and plasmids. METHODS: In this prospective study, we collected blood culture data from hospitalised patients with a suspected bloodstream infection processed from May 1, 2018, to April 30, 2019, and rectal swabs from healthy pregnant women from July 30, 2018, to April 27, 2019, both from three hospitals in Antananarivo, Madagascar; and caeca from farm chickens and surface waters from the Ikopa river, wastewater, and slaughterhouse effluents in the Antananarivo area, Madagascar, from April 9, 2018, to April 30, 2019. All samples were tested for ESBL-E coli. The genomes of all isolates were sequenced using a short-read method on NextSeq 500 and NovaSeq 6000 platforms (Illumina, San Diego, CA, USA) and those carrying plasmid replicons using an additional long-read method on a MinION platform (Oxford Nanopore Technologies, Oxford, UK). We characterised genomes of isolated strains (sequence type, resistance and virulence gene content, and plasmid replicons). We then compared isolates using the variant calling method (single-nucleotide polymorphism). FINDINGS: Data from 1056 blood cultures were collected and 289 pregnant women, 246 chickens, and 28 surface waters were sampled. Of the blood cultures, 18 contained E coli, of which seven (39%) were ESBL. ESBL-E coli was present in samples from 86 (30%) of 289 pregnant women, 140 (57%) of 246 chickens, and 28 (100%) of 28 surface water samples. The wet season (November to April) was associated with higher rates of carriage in humans (odds ratio 3·08 [1·81-5·27]) and chickens (2·79 [1·65-4·81]). Sequencing of 277 non-duplicated isolates (82 from pregnant women, 118 from chickens, and 77 from environmental samples) showed high genetic diversity (90 sequence types identified) with sector-specific genomic features. Single nucleotide polymorphism (SNP) analysis revealed that 169 (61%) of 277 isolates grouped into 44 clusters (two or more isolates) of closely related isolates (<40 SNPs), of which 24 clusters contained isolates from two sectors and five contained isolates from all three sectors. ESBL genes were all blaCTX-M variants (215 [78%] of 277 being blaCTX-M-15) and were located on a plasmid in 113 (41%) of 277 isolates. These ESBL-carrying plasmids were mainly IncF (63 [55%] of 114; one strain carried two plasmids) and IncY (42 [37%] of 114). The F31/36:A4:B1 (n=13) and F-:A-:B53 (n=8) pMLST subtypes, and the IncY plasmids, which were all highly conserved, were observed in isolates of differing genetic backgrounds from all sectors and were transferable in vitro by conjugation. INTERPRETATION: Despite sector-specific population structures, both ESBL-E coli strains and plasmids are circulating among humans, chickens, and the environment in Antananarivo, Madagascar. The Tricycle protocol can be implemented in a low-income country and represents a powerful tool for investigating dissemination of AMR from a One Health perspective. FUNDING: Fondation Mérieux and INSERM, Université Paris Cité.

Prevalence, Risk Factors, and Genetic Characterization of Extended-Spectrum Beta-Lactamase Escherichia coli Isolated From Healthy Pregnant Women in Madagascar.

Antimicrobial resistance is a major public health concern worldwide affecting humans, animals and the environment. However, data is lacking especially in developing countries. Thus, the World Health Organization developed a One-Health surveillance project called Tricycle focusing on the prevalence of ESBL-producing Escherichia coli in humans, animals, and the environment. Here we present the first results of the human community component of Tricycle in Madagascar. From July 2018 to April 2019, rectal swabs from 492 pregnant women from Antananarivo, Mahajanga, Ambatondrazaka, and Toamasina were tested for ESBL-E. coli carriage. Demographic, sociological and environmental risk factors were investigated, and E. coli isolates were characterized (antibiotic susceptibility, resistance and virulence genes, plasmids, and genomic diversity). ESBL-E. coli prevalence carriage in pregnant women was 34% varying from 12% (Toamasina) to 65% (Ambatondrazaka). The main risk factor associated with ESBL-E. coli carriage was the rainy season (OR = 2.9, 95% CI 1.3-5.6, p = 0.009). Whole genome sequencing was performed on 168 isolates from 144 participants. bla CTX-M-15 was the most frequent ESBL gene (86%). One isolate was resistant to carbapenems and carried the bla NDM-5 gene. Most isolates belonged to commensalism associated phylogenetic groups A, B1, and C (90%) and marginally to extra-intestinal virulence associated phylogenetic groups B2, D and F (10%). Multi locus sequence typing showed 67 different sequence types gathered in 17 clonal complexes (STc), the most frequent being STc10/phylogroup A (35%), followed distantly by the emerging STc155/phylogroup B1 (7%), STc38/phylogroup D (4%) and STc131/phylogroup B2 (3%). While a wide diversity of clones has been observed, SNP analysis revealed several genetically close isolates (n = 34/168) which suggests human-to-human transmissions. IncY plasmids were found with an unusual prevalence (23%), all carrying a bla CTX-M-15. Most of them (85%) showed substantial homology (≥85%) suggesting a dissemination of IncY ESBL plasmids in Madagascar. This large-scale study reveals a high prevalence of ESBL-E. coli among pregnant women in four cities in Madagascar associated with warmth and rainfall. It shows the great diversity of E. coli disseminating throughout the country but also transmission of specific clones and spread of plasmids. This highlights the urgent need of public-health interventions to control antibiotic resistance in the country.

A new signaling cascade linking BMP4, BMPR1A, ΔNp73 and NANOG impacts on stem-like human cell properties and patient outcome.

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients' bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients' outcome.

Resurgence of Neisseria meningitidis serogroup W ST-11 (cc11) in Madagascar, 2015-2016.

The resurgence of invasive meningococcal disease caused by Neisseria meningitidis serogroup W with sequence type ST-11 (cc11) was observed in Madagascar in 2015-2016. Three cases were investigated in this study. Molecular characterization of the strains suggests the local transmission of a single genotype that may have been circulating for years.

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens.

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.

Structure-activity relationship of human liver-expressed antimicrobial peptide 2.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1-3 and 2-4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200microM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5microM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.