Linda Hadjadj

Molecular mechanisms of polymyxin resistance: knowns and unknowns

Colistin, also referred to as polymyxin E, is an effective antibiotic against most multidrug-resistant Gram-negative bacteria and is currently used as a last-line drug for treating severe bacterial infections. Colistin resistance has increased gradually for the last few years, and knowledge of its multifaceted mechanisms is expanding. This includes the newly discovered plasmid-mediated colistin resistance gene mcr-1, which has been detected in over 20 countries within 3 months of its first report. We previously reported all of the known mechanisms of polymyxin resistance in our first review in 2014, but an update seems necessary in 2016, considering the significant recent discoveries that have been made in this domain. This review provides an update about what is already known, what is new, and some unresolved questions with respect to colistin resistance.

Real-time quantitative PCR assay with Taqman® probe for rapid detection of MCR-1 plasmid-mediated colistin resistance

Here we report the development of two rapid real-time quantitative PCR assays with TaqMan® probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli) with a calibration curve that was linear from 101 to 108 DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing.

Fortuitous diagnosis of NDM-1-producing Acinetobacter pittii carriage in a patient from France with no recent history of travel.

L’UNAM Université, Université d’Angers, ATOMycA, Inserm Equipe Avenir, CRCNA, Inserm U892, 6299 CNRS – IRIS, CHU, 4 rue Larrey, 49933 Angers cedex, France; Laboratoire de Bactériologie, Institut de Biologie en Santé—PBH, CHU, 4 rue Larrey, 49933 Angers cedex, France; Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), UM 63, CNRS 7278, IRD 198, INSERM 1095, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille Université, Marseille, France; Service de Maladies Infectieuses et Tropicales—CHU, 4 rue Larrey, 49933 Angers cedex, France

Study of mcr-1 Gene-Mediated Colistin Resistance in Enterobacteriaceae Isolated from Humans and Animals in Different Countries

In this study, we aim to characterize the genetic environment of the plasmid-mediated colistin resistance gene mcr-1 in 25 Escherichia coli and seven Klebsiella pneumoniae strains from different countries and continents. Multilocus sequence typing, conjugation experiments, plasmid typing, and the presence and location of the insertion sequence ISApl1 were investigated. Whole genome sequencing of four E. coli was performed to analyse the genetic environment of the mcr-1 gene. Colistin minimum inhibitory concentration of mcr-1 strains varied from 3 to 32 µg/mL. Six E. coli sequence types were detected: ST 4015, ST 3997, ST 10, ST 93, ST 48, and ST 648. IncHI2, IncI2, and IncP plasmid types were predominant and were unrelated to a specific country of origin. ISApl1 was found in 69% of analysed plasmids that were mainly around the mcr-1 gene. Analysis of four closed mcr-1 plasmids revealed the integration of mcr-1 into hotspots. We found that the spread of mcr-1 gene was due to the diffusion of a composite transposon and not to the diffusion of a specific plasmid or a specific bacterial clone. The ease with which the mcr-1 gene integrates into various regions facilitates its dissemination among bacteria and explains its large diffusion all over the world, both in animals and in humans.

New therapy from old drugs: synergistic bactericidal activity of sulfadiazine with colistin against colistin-resistant bacteria, including plasmid-mediated colistin-resistant mcr-1 isolates

The recent emergence of colistin (COL) resistance, particularly mcr-1 plasmid-mediated COL resistance in Gram-negative bacteria, has led to renewed interest in antibiotic combinations to overcome clinical therapeutic impasses. The aim of this study was to evaluate the potential of the synergistic and bactericidal activity of COL in combination with sulphonamide compounds, including sulfadiazine (SDI), sulfamethoxazole (SMX) and trimethoprim/sulfamethoxazole (SXT), as well as trimethoprim (TMP) against clinical COL-resistant bacterial strains, including strains with the plasmid-encoded mcr-1 gene. A collection of 55 COL-resistant and -susceptible strains from different origins (Laos, Thailand and France) was used in this study. Several in vitro methods were used to determine the potential of the synergistic activity of these combinations, including Etest on agar pre-treated plates, the Etest cross method and the chequerboard assay. A time-kill assay was performed to evaluate the potential bactericidal activity of combinations in addition to synergistic activity. Significant synergistic activity was observed with all combinations tested. The combination of COL + SDI presented the highest synergistic effect against the various species of COL-resistant strains (92.7%). For the other combinations, a synergistic effect was also observed but with lower frequency for COL + SMX (33.3%), COL + TMP (47.3%) and COL + SXT (31.5%). Synergy was observed independently of the COL resistance mechanism. These in vitro results suggest that the combination of COL + SDI would appear to be justifiable in patients with multidrug-resistant bacterial infections that cannot be treated with COL monotherapy.

Genome sequence and description of Mannheimia massilioguelmaensis sp. nov.

Strain MG13T sp. nov. is the type strain of Mannheimia massilioguelmaensis, a new species within the genus Mannheimia. This strain was isolated from the exudate of a skin lesion of an Algerian man. Mannheimia massilioguelmaensis is a Gram-negative, facultative anaerobic rod, member of the family Pasteurellaceae. Here we describe this organism, together with the complete genome sequence and annotation. The 2 186 813 bp long genome contains 2048 protein-coding and 55 RNA genes, including eight rRNA genes.

Characterization of bla OXA-538 , a new variant of bla OXA-48 in Shewanella xiamenensis isolated from River water in Algeria

OBJECTIVES In this study, the presence of carbapenemase genes in Shewanella xiamenensis strains isolated from river water in Béjaïa, Algeria, was investigated. METHODS Four isolates of S. xiamenensis were isolated from water from Soummam River. The isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and sequencing of the 16S rRNA and gyrB genes. Isolates were subjected to antimicrobial susceptibility testing. Carbapenemase production was screened using phenotypic tests. PCR and sequencing were used to identify carbapenemase genes in the isolates. The genetic context of the blaOXA-48-like gene was investigated by sequencing the whole genome of strain AS58. RESULTS All four S. xiamenensis strains harboured blaOXA-48-like genes. They exhibited different resistance patterns and had imipenem minimum inhibitory concentrations (MICs) of ≥0.5mg/L. Sequencing of blaOXA-48-like genes from the S. xiamenensis isolates showed that two strains harboured blaOXA-181, one strain harboured blaOXA-199 and one strain exhibited a new variant of the blaOXA-48-like gene, named blaOXA-538. This new variant shared 98% nucleotide identity with blaOXA-162, with three amino acid changes (G201A, A213G and I219F). Conjugation assays with Escherichia coli J53 recipient were performed but no transconjugants were obtained. Analysis of the genome of AS58 Touati strain confirmed the chromosomal location of the blaOXA-538 gene. CONCLUSION This study showed that environmental water holds a diversity of S. xiamenensis strains harbouring blaOXA-48-like genes and may play an important role in the dissemination and spread of these genes from the environment to humans.

Genome sequence and description of Desnuesiella massiliensis gen. nov., sp. nov. a new member of family Clostridiaceae

Desnuesiella massiliensis, strain MT10T gen. nov., sp. nov. is a newly proposed genus within the family Clostridiaceae, isolated from the digestive microbiota of a child suffering from kwashiorkor. Desnuesiella massiliensis is a facultatively anaerobic, Gram-positive rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5 503 196-bp long genome (one chromosome but no plasmid) contains 5227 protein-coding and 81 RNA genes, including 14 rRNA genes.

Role of Institut Hospitalo-Universitaire Méditerranée Infection in the surveillance of resistance to antibiotics and training of students in the Mediterranean basin and in African countries

Surveillance of antibiotic resistance has become a public global concern after the rapid worldwide dissemination of several antibiotic resistance genes. Here we report the role of the Institut Hospitalo-Universitaire Méditerranée Infection created in 2011 in the identification and description of multidrug-resistant bacteria thanks to collaborations and training of students from the Mediterranean basin and from African countries. Since the creation of the institute, 95 students and researchers have come from 19 different countries from these areas to characterize 6359 bacterial isolates from 7280 samples from humans (64%), animals (28%) and the environment (8%). Most bacterial isolates studied were Gram-negative bacteria (n = 5588; 87.9%), mostly from Algeria (n = 4190), Lebanon (n = 946), Greece (n = 610), Saudi Arabia (n = 299) and Senegal (n = 278). Antibiotic resistance was diversified with the detection and characterization of extended-spectrum β-lactamases, carbapenemases and resistance to colistin, vancomycin and methicillin. All those studies led to 97 indexed international scientific papers. Over the last 6 years, our institute has created a huge network of collaborations by training students that plays a major role in the surveillance of resistance to antibiotics in these countries.

Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Clinical_Isolates: Algeria.

This study investigates the molecular mechanisms of colistin and carbapenem resistance in Klebsiella pneumoniae ST101 strains. The three K. pneumoniae carried blaCTX-M-15, blaTEM-183, and blaSHV-106 genes and two coharbored blaOXA-48. As for colistin resistance, the isolates had amino acid substitutions in PmrA/B and a truncated mgrB gene in one isolate.