Anti-infectives – Antimicrobial agents and resistance, monitoring and therapeutic strategies
UR D-258, Microbes, Evolution, Phylogénie et Infection (MEPHI)
Aix-Marseille Université (AMU)
Team leaders :
ROLAIN Jean-Marc (PU-PH, AMU/AP-HM)
DIENE Seydina (MCF, AMU, HDR)
Members
BARON Sophie
BITTAR Fadi
COMMOT Muriel
HADJADJ Linda
MERHEJ Vicky
PAGNIER Isabelle
Introduction
The emergence and spread of antibiotic resistance (AR) in bacteria are nowadays a major public health problem worldwide. Hospital-acquired infections caused by multidrug-resistant bacteria (MDR) have not only led to increased mortality, morbidity, and treatment costs, but also continue to endanger the lives of immunocompromised patients in hospitals. As reported the CDC (Centers for Diseases Control and Prevention) estimates that 50% of antibiotics (used to treat bacterial infections) administered in hospitals are inappropriate or unnecessary. Of course, the uncontrolled and abusive use of antibiotics in different ecosystems such as in humans, animals, or in agriculture, has greatly contributed to the wide spread of this bacterial resistance. For example, there is a staggering antibiotic use in agriculture since about 70% of the global consumption of antibiotics is used in animal and fish farming and to spray on crops. More than 130’000 tons were used on farmed animals in 2013, with this amount expected to rise by 53% by 2030 because of the rising of meat consumption. However, recent studies have shown that antibiotic resistance is a natural phenomenon that can emerge from ancient and/or environmental sources. Thus, in the face of this global concern, several studies have been reported with important recommendations to conduct epidemiological, molecular, and genomic studies to control the spread and increase of antibiotic resistance. This was the case in the report the recent “World Antibiotic Awareness Week” event in November 2017, WHO (World Health Organization), and partners who reached out to the general public, health professionals, governments, farmers, veterinarians, food and feed industries on the need to act against antibiotic resistance and what kinds of steps we can take. Nowadays, living in a global economy, the movement of people, goods and foods can have a massive and very rapid impact on AR dissemination over a wide area. In addition to that, animals such as wide birds including migratory birds, able to cover several countries, have been reported to vehicle antimicrobial resistance determinants and thus can greatly participate to the AR dissemination.
However, during the last 10 years, we have witnessed the emergence and development of new high-throughput sequencing technologies coinciding with an exponential increase in the number of sequenced bacterial genomes. It is in this perspective that our research activities are mainly articulated on four axes, namely:
Axis 1: Discovery of new emerging pathogens and new mechanisms of resistance to antimicrobial agents – Cross-sectional studies in hospitals, in humans, in agriculture (animals in particular) and in the environment.
The objective of this research axis is to isolate and describe new and/or emerging microorganisms associated with humans, the environment and animals and to describe the molecular basis of antimicrobial resistance in these microorganisms. The objective of this task is to have both an overview of the repertoire of microorganisms, their antimicrobial resistance profile, but also to describe the different resistance mechanisms that bacteria develop against antimicrobial agents. Thus, this research axis will include the following projects :
- Isolation new and/or multi-resistant microorganisms (bacteria, parasites, viruses) from different samples by high throughput Culturomics methods and patented selective media (LBJMR medium) (FB, IP, SB). Indeed, in this task we plan to collect samples from frequented area such as parks, beaches, and markets. These samples will be potential sources and/or reservoirs (soils, waters, fecal samples of urban animal, birds, chickens) of bacterial pathogens of clinical interest. Also, veterinary, butcheries, and farms will be visited in order to perform samplings from these environments including workers in these settings. The presence of MDR bacteria will be investigated in these different sources by massive bacterial cultures using selective culture media such as the LBJMR that was created and patented by our team and other selective media such as MacConkey supplemented with imipenem or ertapenem. In our laboratory, we already have in our disposal various collections of samples from human, especially from human microbiota, from animal microbiota, from the environment (wastewaters from hospital, from frequented parks, and from beaches). Our preliminary results reported that some animals like urban birds are sources of MDR bacteria especially extended-spectrum-β-lactamases producing bacteria and those birds may participate to zoonotic transmission since some bacterial clone of Escherichia coli already responsible of human infections have been identified in the studied urban bird fecal samples.
- Characterization of the antimicrobial susceptibility of isolated bacteria from these sources by development and testing of specific phenotypic assays (FB, IP, SE, SB, LH). Indeed, the culturomics approach and selective bacterial cultures, all isolates will be subjected to molecular identification using the MALDI-TOF MS, a rapid and powerful tool for bacterial identification and their full antibiotic resistance phenotype will be determined using a panel antibiotics including amoxicillin (AMX); amoxicillin/clavulanic acid (AMC); ticarcillin; ticarcillin/clavulanic acid(TIM); piperacillin/tazobactam (TZP); ceftriaxone (CRO); ceftazidime (CAZ); cefepime (FEP); aztreonam (AZT); imipenem (IPM); ertapenem (ERT); amikacin (AK); gentamicin (GEN); tobramycin (TOB), ciprofloxacin (CIP); trimethoprim/sulfamethoxazole (SXT); fosfomycin (FF), nitrofurans (F), doxycycline (DO); rifampicin (RIF) and colistin (CS).
- Genomes sequencing using HST methods including Illumina MiSeq and Nanopore sequencers will be performed in order to investigate the molecular basis of their resistance to antimicrobials. Indeed, raw sequencing data will be firstly subjected to quality control check using bioinformatic tool such as fastqc program, in order to verify the required among of sequence data for each sequenced genome. After that, genome assembly of MiSeq sequence data will be performed using several assemblers including, A5_MiSeq, Edena, SPades. Data from Nanopore sequencing will be assembled using Canu program. From these analyses, the best assembly result for each genome will be kept for further analyses. Indeed, when genomes were properly assembled, they were then subjected to genome annotation using a developed pipeline in our team that includes (i) “Prokka” annotation pipeline performing the ORFing (gene identification) and the prediction of protein function, (ii) “PlasmidSeeker” program allowing to identify plasmid sequence from the assembled genomes, (iii) “ABRicate” program allowing the identify the full resistome of a genome by “BlastN” against the three main Antibiotic resistance genes databases (“ARG-ANNOT”, “ResFinder”, and “CARD”). Comparative genomics will be performed using “Roary” pipeline that allow to identify the Coregenome, Pangenome, and accessory genome of a set of genomes. Clonal relationship between isolates will be investigated using “Parsnp” and “gMLTS” programs, capable to evaluate the number of single nucleotide polymorphisms (SNP) between genomes. Specific analyses such as phylogenetic tree analysis, genome alignment, circos analysis will be performed on specific bacterial genomes to more described interesting and particular isolates. It is important to note that these approaches will be combined with innovative methods (functional genomics, transpositional mutagenesis, knock-in/knock-out, silencing) to investigate deeply atypical strains with particular resistance phenotypes (SMD, SB, LH).
- Based on collected sequence data, massive bioinformatics analyses will be conducted on these bacterial genomes to figure out the modes of acquisition of exogenous sequences (including resistance genes) but also the defense mechanisms against exogenous DNA sequences through CRISPR-Cas systems. Thanks, to our bioinformatic expertise, we plan to conduct works to investigate the involvement of CRISPR-Cas systems in the acquisition of resistance genes especially in Gram-negative isolates (e.g., Klebsiella pneumoniae) responsible of hospital-acquired infections. Our previous works have demonstrated in Gram-positive bacteria, especially in Enterococcus species (1591 E. faecalis genomes vs, 1981 E. feacium genomes) a significant association of the presence of these CRISPR-Cas systems and the absence of vancomycin resistance genes in E. faecalis species as compared with E. feacium species in which the massive presence of vancomycin resistance genes is associated with the absence of these defense systems. We can note that, this approach could lead to new therapeutic approaches or strategies against infections by certain bacterial species (SMD, VM, SB).
- Investigation of the diversity, origin, and multifunctionality of β-lactamases, in particular metallo-β-lactamases (MBLs) in different domains of life including, Bacteria, Archaea and related microorganisms such as Nanoarchaeota and Asgard, Eukaryotes (humans), Giant Viruses, and Candidate phyla radiations (SMD, JMR, FB, SB, LH). Our recent publications have been able to demonstrate the existence of these MBLs in these different organisms with multiple activities including β-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, and so many other enzymatic activities.
Axis 2: Discovery of new antimicrobial agents
Faced with the emergence of antibiotic-resistant pathogens and the lack of development of new antimicrobial agents, there is an urgent need to discover and develop new classes of antimicrobial agents with novel bacterial inhibitory mechanisms that could replace or be combined with traditional antibiotics to fight multi-resistant pathogens. Most antimicrobials are secondary metabolites produced by certain microorganisms (Actinobacteria, Firmicutes and fungi) and represent inexhaustible sources of natural products for the future (bacteriocins, non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS)). Although the majority of antimicrobials have been discovered to date among environmental isolates, natural antimicrobial agents from human gut microorganisms have never been explored. Through the reuse of the bacterial culture strategy called « Culturomics », IHU has isolated more than 845 bacterial species (including 121 new bacterial species) and 59 fungal species from human microbiota from different geographical regions. Our project consists in discovering new antimicrobial agents from bacteria and fungi of interest selected from this collection of human microorganisms (about 200 strains to date). This project has 5 axes:
- The genome sequencing of these species as well as the new microorganisms isolated from Project 1 (SMD, VM, SB, LH,).
- To develop a pipeline including a comprehensive database of genes of interest for annotation and automatic recognition of secondary metabolites encoding resistance genes in these genomes (SMD, SB, VM, LH).
- In vitro search of putative secondary metabolite producing bacteria, for antimicrobial activity on a collection of contemporary multidrug resistant bacteria and fungi (VM, FB, LH). Indeed, we develop innovative culture methods to test any antimicrobial activity of any organisms on a collection of bacteria including MDR bacteria.
- Use transcriptomic and proteomic approaches to identify new natural products and their mode of action/synergy and structure determination (SMD, SB, VM, LH). This task will conduct in collaboration with Dr Saïd AZZA and ARSMTRONG Nicholas, responsible of the proteomic platform of our institute.
- Evaluate their toxicity on mammalian cells (SE) and animals in order to demonstrate their efficacy in vivo on experimentally infected animal models (in collaboration with Pr Fabienne Bregeon).
Development of new tools for epidemiology, diagnosis and therapeutic strategies
The first task will be to continue to develop specific and new tools for the detection and diagnosis of infections due to emerging and multidrug resistant microorganisms, with a specific effort for parasites, protozoa and intracellular microorganisms associated with protozoa (FB, IP). Among the different tools that will be developed, we will implement a unique tool and strategy for automatic recognition of antibiotic susceptibility of bacteria using Anti-Logic software that will be integrated into a larger project to develop a hub for the institute (SE, SB) (see below in axis 4).
The second task of the team will be to build up a unique biobank of all available antimicrobials (antibiotics, antifungals and antiparasitic agents), including old compounds that are no longer marketed in France and that will be tested in vitro against multidrug resistant human pathogens (SE, IP, FB) and that could be used in the institute to treat specific infectious diseases with these compounds. The objective is both to be able to test these compounds in vitro in specific clinical situations and to be able to use them in the Institute’s infectious disease units. Several of these compounds are no longer available in France or are not marketed by pharmaceutical companies, so we will have to develop partnerships with pharmaceutical companies for their production in coordination with regulatory agencies.
The third task will be to use high-throughput in vitro testing methods to screen all clinically approved and marketed drugs (about 2,000 compounds) for antimicrobial activities to reposition drugs in the infectious disease field (SE, FB, IP). Such strategy has recently been used successfully, for example, to show that teicoplanin, an antibiotic used clinically for its activity against Gram-positive bacteria, could inhibit Ebola viruses. Similarly, it was shown that ivermectin, an antiparasitic compound, could inhibit the replication of Chikungunya virus. In this task, new drug activities against microorganisms will be patented if new indications are not protected in the field of infectious diseases.
Finally, the team will continue to develop new therapeutic strategies to fight infections due to MDR pathogens (combination of drugs), as well as innovative treatments for tropical diseases (parasites, protozoa) and pediculosis. Again, patents will be filed for a new combination of drugs with specific clinical indications.
Epidemiological and molecular surveillance of antimicrobial resistance (human, animal and environment)
The surveillance tools for infectious diseases and antibiotic resistance will be extended step by step to all partner laboratories in the PACA region, which now covers about 80% of the clinical microbiology laboratories in this region. The team will continue to develop its partnerships with Mediterranean countries (Tunisia, Morocco, Algeria, Libya, Egypt, Spain, Italy, Greece…) and will strengthen its new networks with Africa (Senegal, Mali, Nigeria, DRC, Gabon, Cameroon, Djibouti), the Middle East (Lebanon, Israel, Saudi Arabia, Qatar), Asia (Thailand, Laos, Vietnam, China, Taiwan) and Australia for epidemiological research and molecular surveillance of antimicrobial resistance in humans, but also in animals and in the environment For this purpose, the three main surveillance tools (EPIMIC, BALYSES and MARSS) will be progressively extended to the partners of the two structured projects REMEDIER and GIRAFE (Algeria, Senegal and Mali), then to other partners.
The team will also continue to participate in the detection and surveillance of antibiotic resistance in travelers, particularly in pilgrims. Surveillance of bacterial clones, especially multidrug resistant bacteria, will be implemented using MALDI-TOF typing as a routine tool (MDS, SE, SB, LH). Specific resistance, i.e., new and/or emerging resistance genes, will be systematically studied by developing and implementing phenotypic and genotypic tools in the institute.
In this line of research, we will also carry out work on the implication of the use of pesticides in the selection and emergence of multi-resistant bacteria through co-resistance phenomena. Indeed, we will carry out more targeted work on pesticides such as glyphosate, which is already patented as an antibiotic and is therefore the most widely used antibiotic in the world today. Our recent work on this topic has provided evidence of the involvement of glyphosate in the selection of β-lactam resistant Enterobacteriaceae such as Escherichia coli or Klebsiella pneumoniae.
Finally, we will develop a single hub (data concentrator) for the institute, capable of intervening, analyzing and linking all the activities of the institute in order to improve the workflow in the clinical microbiology laboratory and to specifically detect all abnormal events associated with infectious diseases in real time including automatic outbreak detection and antibiotic resistance.
Expertise
The team includes experts in the field of infectious diseases dedicated to the description and study of new and emerging pathogens in the context of antimicrobial resistance.
Dr. Fadi Bittar (PhD, Associate Professor at the Faculty of Pharmacy) is now specialized in the detection, isolation and description of parasites, protozoa, fungi, but also candidate phyla radiations (CPR). He will participate in the development of specific and sensitive tools for the diagnosis and monitoring of antimicrobial resistance (antiparasitic and antifungal compounds).
Dr. Isabelle Pagnier (PharmD, PhD, HDR, and Associate Professor at the Faculty of Pharmacy) has expertise in intracellular microorganisms (bacteria, viruses) associated with protozoa and in electron microscopy. These microorganisms are often resistant to antimicrobials and will be studied and characterized by Dr. Pagnier to determine their molecular basis for antimicrobial resistance.
Dr. Seydina M. Diene (PhD, HDR, Associate Professor at the Faculty of Pharmacy) is an expert in bioinformatics and genomic analysis of bacteria in the context of antibiotic resistance. He will be involved in all tasks of the team related to genomics of all microorganisms (bacteria, viruses, fungi, parasites). Dr. Seydina M. Diene also has extensive experience in the evolution of bacterial genomes and genetic manipulation of bacteria.
Dr. Sophie Edouard (PharmD, PhD, Associate Professor at the Faculty of Medicine) is a clinical microbiologist with extensive experience in Culturomics and metagenomic analysis of human gut microbiota in patients treated with antimicrobials and/or patients with specific infectious diseases. She is also involved with Prof. JM Rolain in the management of routine analysis of clinical samples and antibiotic resistance susceptibility testing and surveillance at the Institute.
Dr Vicky Merhej (PharmD, PhD, Associate Professor at the Faculty of Medicine) is an expert in the massive analysis of bacterial genomic sequences but also in the research of secondary metabolites in microorganisms. She is also an expert in bioinformatics, particularly in the creation of sequence databases, both of antibiotic resistance genes and of NRPS/PKS databases, the gene clusters responsible for the synthesis of secondary metabolites. She will also participate in the development of a more fundamental understanding of the mechanisms of transfer of mobile genetic elements between different microorganisms (transposons, integrons, ICE plasmids….).