CROSS-REFERENCE TO PRIOR APPLICATIONS
This is a divisional of application Ser. No. 11/794,907 filed Jul. 9, 2007, which is a National Stage Application of PCT/FR2006/050109 filed Feb. 9, 2006, and claims the benefit of French Application Nos. 0550394 filed Feb. 10, 2005 and 0553049 filed Oct. 7, 2005. The entire disclosures of the prior applications are hereby incorporated by reference herein in their entirety.
The field of the invention is that of microbiological analysis by means of biochemistry, and in particular the detection and identification of microorganisms, for instance of bacteria or yeasts.
Bacterial resistance to antibiotics is a major public health problem. The resistance of infectious microorganisms to a treatment has developed at the same time as anti-infectious molecules and today represents a major obstacle in therapeutics. This resistance is responsible for many problems, including difficulties in detection in the laboratory, limited treatment options and a deleterious impact on clinical outcome.
In particular, the rapid and irrepressible increase in the resistance of pathogenic bacteria, over the last 20 years, represents one of the major current problems in medicine. Infections caused by these organisms are responsible for extended periods of hospitalization and are associated with high morbidity and mortality rates, following therapeutic failures.
Several resistance mechanisms can be involved simultaneously in a bacterial strain. They are generally classified in 3 categories: deficient penetration of the antibiotic into the bacterium, inactivation or excretion of the antibiotic by bacterial enzymatic systems, and lack of affinity between the bacterial target and the antibiotic.
Enzymatic inactivation is the most common mechanism of acquired resistance in terms of number of species and of antibiotics involved. Thus, chromosomal class C cephalosporinases today constitute one of the predominant resistance mechanisms of gram-negative bacteria, the bacteria expressing such enzymes being resistant to cephalosporins. Similarly, β-lactamases are enzymes expressed by certain bacteria, capable of hydrolyzing the C—N bond of the β-lactame ring, the basic structure of antibiotics of the β-lactamine family, so as to give a microbiologically inactive product. Several β-lactamase inhibitors (BLIs), such as clavulanic acid (CA), tazobactam and sulbactam, have been developed in order to increase the antimicrobial activity and broaden the spectrum of the β-lactamines which are associated therewith. They act as a suicide subject for β-lactamases, and prevent enzymatic degradation of the antibiotics and allow them to become effective against bacteria that were initially resistant. However, by virtue of the persistent exposure of strains to antibiotic pressure, the bacteria express their ability to adapt through the continuous and dynamic production of β-lactamases, which evolves at the same time as the development of new molecules.
Gram-negative bacteria which produce high-level chromosome class C cephalosporinases (reference is made to HL Case bacteria), and also gram-negative bacteria which produce extended-spectrum β-lactamase (reference is then made to ESBL bacteria) have, as a result, become an increasing threat, in particular because the number of bacterial species concerned is increasing. HL Case and ESBL bacteria are resistant to treatments based on 1st- and 2nd-generation penicillins and cephalosporines, but also on 3rd-generation cephalosporines (C3G) (cefotaxim CTX, ceftazidime CAZ, cefpodoxime CPD, ceftriaxone CRO) and monobactams (aztreonam ATM). On the other hand, 7α-methoxycephalosporins (cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem, meropenem, ertapenem) generally conserve their activity. ESBLs are inhibited by β-lactamase inhibitors (BLIs), which makes it possible to differentiate them from other cephalosporinases.
These bacteria thus most commonly simultaneously express resistances to several treatments, which poses difficulties in setting up a relevant treatment and avoiding therapeutic failures. An Escherichia coli bacterium can thus be HL Case and ESBL. In addition, since ESBL-positive enterobacteria have a tendency to disseminate the resistance by clonal transmission of strains or conjugative plasma transfer, they represent a problem in terms of controlling infections. In most studies, Escherichia coli and Klebsiella pneumoniae remain the most common ESBL-producing species. However, over the last few years, ESBLs have greatly broadened their panel of host species. In fact, many species of enterobacteria and of nonfermenting gram-negative bacilli (such as Pseudomonas aeruginosa) have also been reported to ESBL producers.
In addition to these ESBL bacteria, mention may also be made of Staphylococcus aureus bacteria, which are also pathogenic bacteria that develop many mechanisms of resistance, such as resistance to methicillin, penicillin, tetracycline, erythromycin, or vancomycin. Enterococcus faecium is another multiresistant bacterium found in the hospital environment, which can be resistant to penicillin, vancomycin and linezolide. Mycobacterium tuberculosis is commonly resistant to isoniazid and to rifampicin. Other pathogens offer certain resistances, such as Salmonella, Campylobacter and Streptococcus.
It therefore becomes essential, from a public health point of view, to be able to identify such microorganisms, and such resistance mechanisms, as rapidly as possible.
In general, the search for microorganisms resistant to a treatment is carried out according to the following steps:
1. Taking a biological sample that may contain said microorganisms;
2. Seeding and incubating a culture medium (18 to 48 h) in order to induce exponential growth of the microorganisms;
3. Pinpointing, on the culture media, colonies of potentially significant microorganisms;
4. Characterizing the microorganism species;
5. Identifying the mechanisms of resistance of the microorganisms analyzed, their biological significance and, optionally, the appropriate therapy.
This succession of steps involves a considerable amount of time between taking the sample that may contain microorganisms and prescribing a treatment that is appropriate for the patient. Furthermore, the user must generally perform steps for transferring microorganims from a first medium to a second medium manually, which can induce problems, in particular, of contamination, but also risks to the handler\'s health.
By way of example, in order to detect the presence of broad-spectrum beta-lactamases (ESBLs) in strains of Escherichia coli and Klebsiella pneumoniae, use may be made of a diffusion technique as described in the publication by Jacoby & Han (J Clin Microbial. 34(4): 908-11, 1996), which does not however give any information regarding the identification of the strains tested: it is possible to determine whether or not the bacterium is a ESBL-producing bacterium, but it is not possible to distinguish whether such a bacterium is an Escherichia coli or a Klebsiella pneumoniae.
Metabolic substrates are also used for detecting the presence of ESBLs or HL cases. In this respect, AES laboratories proposes a medium in a biplate combining a Drigalski medium with cefotaxim and a MacConkey medium with ceftazidime. The Drigalski and MacConkey media make it possible to reveal lactose acidification, a metabolism which is present in a very large number of enterobacterial species. However, such a medium only makes it possible to distinguish resistant bacteria from non-resistant bacteria, and does not make it possible to distinguish bacteria expressing a ESBL from those expressing an HL Case. Neither does this medium make it possible to identify specific bacterial species, nor does it make it possible, for example, to discriminate between E. coli bacteria and K. pneumoniae bacteria.
In the case of the detection of resistance mechanisms other than ESBL, mention may be made of patent application EP0954560, which relates to the search for Vancomycin-resistant enterococcal, by combining Vancomycin with a chromogenic media that reveals two enzymatic activities (β-glucosidase and pyrrolidonyl arylamidase). However, this chromogenic medium makes it possible to determine only whether or not the vancomycin-resistant strains belong to the Enterococcus genus, but does not make it possible to identify the species or the resistance mechanisms involved, in particular if it is a question of an acquired or wild-type resistance.
Thus, the characterization of a species of microorganism, and then the identification of its resistance to a treatment, is long and laborious. If the laboratory gives the clinician a positive screen, whereas the isolate is in fact free of resistant microorganisms, this can lead to needless and inappropriate treatment. Conversely, not communicating a positive screen, which is subsequently confirmed, delays the setting of the isolation of the patient (and possibly an appropriate therapy) by one day. This shows the need for a rapid and reliable confirmation test.
The present invention therefore proposes to improve the prior art by providing a novel diagnostic tool which allows a gain in time, in reliability and in relevance with respect to the therapy implemented. Our invention makes it possible, in a single step, to identify the species of microorganisms present in a sample, and to determine their mechanism of resistance in order to propose a treatment appropriate to each patient. This invention is particularly suitable for discriminating various species of microorganisms, which have various mechanisms of resistance to various treatments, but all of which may be present in the same sample.
Before going any further in the disclosure of the invention, the following definitions are given in order to facilitate understanding of the invention:
The term “culture medium” is intended to mean a medium comprising all the elements required for the survival and/or the growth of microorganisms. The culture medium according to the invention may contain any possible additives, for instance: peptones, one or more growth factors, carbohydrates, one or more selective agents, buffers, one or more gelling agents, etc. This culture medium may be in liquid form or in gel form which is ready to use, i.e. ready for seeding in a tube or flask or on a Petri plate.
For the purpose of the present invention, the term “microorganism” covers gram-positive or gram-negative bacteria, yeasts and, more generally, organisms that are generally unicellular, invisible to the naked eye, which can be multiplied and handled in the laboratory.
By way of gram-negative bacteria, mention may be made of bacteria of the following genres: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, and Legionella.
By way of gram-positive bacteria, mention may be made of bacteria of the following genre: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria.
By way of yeasts, mention may be made of yeasts of the following genre: Candida, Cryptococcus, Saccharomyces and Trichosporon.
The term “biological sample” is intended to mean a clinical sample, derived from a specimen of biological fluid, or a food sample, derived from any type of food. This sample may thus be liquid or solid and mention may be made, in the nonlimiting manner, of a clinical blood, plasma, urine or faeces sample, nose, throat, skin, wound or cephalospinal fluid specimens, a food sample from water, from drinks such as milk or a fruit juice; from yoghurt, from meat, from eggs, from vegetables, from mayonnaise, from cheese; from fish, etc., a food sample derived from a feed intended for animals, such as, in particular, a sample derived from animal meals.
The term “mechanism of resistance” is intended to mean any type of device which allows a microorganism to render a treatment partially or completely ineffective on said microorganism, guaranteeing its survival. The mechanisms of resistance are generally divided up into three categories: deficient penetration of the antibiotic into the bacterium, inactivation or excretion of the antibiotic by means of bacterial enzymatic systems, and lack of affinity between the bacterial target and the antibiotic.
By way of indication, mention may in particular be made of mechanisms of resistance related to the expression of an enzyme belonging to the broad-spectrum β-lactamase group; of an enzyme belonging to the chromosomal high level class C cephalosporinase group; mechanisms of resistance to glycopeptides, preferably developed by bacteria belonging to the Enterococcus genus.
Mention will also be made of mechanisms of resistance to methicillin, penicillin, tetracycline, erythromycin, or vancomycin when the microorganism is a Staphylococcus aureus bacterium.
Mention will also be made of mechanisms of resistance to penicillin, vancomycin and linezolide when the microorganism is an Enterococcus faecium bacterium.
Mention will also be made of mechanisms of resistance to amphotericin B or to antifungals of the azole family when the microorganism is a yeast.
Finally, mention will be made of mechanisms of resistance to isoniazid and to rifampicin when the microorganism is a Mycobacterium tuberculosis bacterium.