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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the present invention relates to a method for detecting and identifying carbapenem-resistant bacteria. More specifically, the method according to the present invention aims to detect bacteria having an enzymatic resistance to carbapenems.
• beta-lactam antibiotics such as penicillins and cephalosporins
• antibiotics are then replaced by other broad-spectrum antimicrobials.
• carbapenems have gained prominence, particularly for treating hospitalized patients. Carbapenems are active against most Gram-positive and Gram-negative aerobic bacteria as well as some anaerobic bacteria.
• the bacteria concerned are, in a non-exhaustive manner, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter sp., Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Providencia rettgeri, Pseudomonas putida, Stenotrophomonas maltophilia, Acinetobacter baumanii, Comamonas sp., Aeromonas sp. Morganella morganii, Enterococcus sp., Proteus mirabilis, Salmonella senftenberg, Serratia marcescens, Salmonella typhimurium etc.
• the reduced susceptibility to carbapenems may be due to:
• carbapenemases are combined with changes in cell wall permeability (impermeability resistance) and / or active efflux of antibiotics (Pages et al., 2009; PloS ONE, 4 (3)); and or • the existence of carbapenem-degrading enzymes, called carbapenemases.
• the carbapenemase genes are likely to be present in chromosomes and / or in plasmids. Because of this presence in the form of plasmids, these enzymatic type resistances are likely to spread very significantly and therefore have a major risk in terms of epidemiology. Resistance due to membrane impermeability to carbapenems is not likely to spread, it is recommended, in the context of diagnosis, monitoring of carriage or hygiene, to be able to distinguish between the two types of resistance .
• the Applicant has shown that it is possible to improve the direct distinction, in particular in a single detection device, between the enzymatic resistance and the resistance by other mechanisms, among which impermeability, by inhibition of the strains exhibiting impermeability or other non-enzymatic resistance mechanism, without affecting the growth and detection of resistant strains by carbapenemase production.
• the Applicant has surprisingly observed an impact of cloxacillin, with or without PAbetaN, on carbapenem-resistant strains that do not produce carbapenemase and do not produce AmpC.
• the present invention relates to a method for detecting and / or identifying, in a biological sample, bacteria exhibiting carbapenem resistance, comprising the steps of:
• reaction medium comprising at least one antibiotic of the class of carbapenems, and cloxacillin
• the medium used in step a) also comprises phenylalanine-arginine-beta-naphthylamide (PAbetaN).
• the Applicant has surprisingly shown that the addition of cloxacillin or a combination of cloxacillin and PAbetaN in a chromogenic or non-chromogenic medium comprising carbapenems makes it possible to improve the sensitivity and the specificity of the detection of bacteria.
• carbapenem-resistant by producing carbapenemases and more specifically identifying carbapenemase-producing strains.
• the method according to the invention makes it possible to differentiate, among the carbapenem-resistant bacterial strains, the carbapenemase-producing strains, the strains exhibiting resistance by impermeability or another non-enzymatic resistance mechanism.
• the hygiene measures necessary to prevent the transmission of strains with enzymatic resistance can be implemented without delay.
• the present invention corresponds to a method for detecting and / or identifying, in a biological sample, bacteria exhibiting carbapenem resistance by producing carbapenemase, comprising the steps of:
• reaction medium comprising at least one antibiotic of the class of carbapenems, and cloxacillin
• the medium used in step a) also comprises phenylalanine-arginine-beta-naphthylamide (PAbetaN).
• PAbetaN phenylalanine-arginine-beta-naphthylamide
• the present invention corresponds to a method for detecting and / or identifying, in a biological sample, bacteria exhibiting carbapenem resistance by producing carbapenemase, comprising the steps of: a) contacting said sample with a reaction medium comprising at least one chromogenic substrate, at least one antibiotic of the carbapenem class, and cloxacillin;
• the medium used in step a) also comprises phenylalanine-arginine-beta-naphthylamide (PAbetaN).
• PAbetaN phenylalanine-arginine-beta-naphthylamide
• Biological sample means a small or small amount isolated from an entity for analysis. It can be a clinical sample, human or animal, from a biological fluid sample, or a food sample, from any type of food, or a sample from the food production or processing environment. This sample can be liquid or solid. There may be mentioned in a nonlimiting manner, a clinical sample of whole blood, serum, plasma, urine, faeces, nose samples, throats, skin, wounds, cerebrospinal fluid, a food sample of water, beverages such as milk, fruit juice, yogurt, meat, eggs, vegetables, mayonnaise, cheese, fish ..., a food sample from a feed intended for animals, such as in particular a sample derived from animal meal, a sample for surface control or water. This sample can be used as it is or, prior to the analysis, undergo an enrichment-type preparation, dilution, extraction, concentration, purification, according to methods known to those skilled in the art.
• reaction medium a medium comprising all the elements necessary for the expression of a metabolism and / or the growth of microorganisms.
• the reaction medium may be solid, semi-solid or liquid.
• solid medium is meant for example a gelled medium.
• Agar is the traditional gelling agent in microbiology for the cultivation of microorganisms, but it is possible to use gelatin, agarose or other natural or artificial gelling agents.
• a number of preparations are commercially available, such as Columbia agar, Trypcase-soy agar, Mac Conkey agar, Mueller Hinton agar or more generally those described in the Handbook of Microbiological Media (CRC Press).
• the reaction medium may comprise one or more elements in combination, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, etc.
• the medium may also comprise a dye.
• dye of Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green , one or more metabolic indicators, one or more metabolic regulators ...
• the reaction medium may be a revelation medium or a culture and revelation medium.
• the culture of the microorganisms is carried out before seeding, and in the second case, the detection and / or identification medium also constitutes the culture medium.
• Those skilled in the art can also use a bi-box, which makes it easy to compare two media, comprising different substrates or different selective mixtures, on which a same biological sample has been deposited.
• the reaction medium may comprise one or more selective agents.
• selective agent is meant any compound capable of preventing or slowing the growth of a microorganism other than the target microorganism. Without being limiting, a concentration of between 0.01 mg / l and 5 g / l is particularly suitable for the present invention.
• antibiotics we mean any compound that can prevent or slow down the growth of a bacteria. They belong especially to the beta-lactam groups, glycopeptides, aminoglycosides, polypeptides, sulfonamides, quinolones.
• antifungal any compound that can prevent or slow the growth of yeast or mold.
• amphotericin B fluconazole, itraconazole, voriconazole, cycloheximide.
• the carbapenems used in the medium used in step a) are stable in a reaction medium.
• they are selected from: meropenem, ertapenem, doripenem, faropenem, thienamycin, biapenem, lenapenem, panipenem, razupenem, tomopenem, tebipenem, sulopenem, beta-methyl carbapenems described by Choi et al. . in the US application 2010/0160284 A1 More preferentially, they are selected from: meropenem, ertapenem, doripenem and faropenem.
• the carbapenem concentrations are between 0.05 and 32 mg / L.
• the concentrations of carbapenems are between 2 and 32 mg / L for faropenem, between 0.05 and 2 mg / L for doripenem, between 0.05 and 2 mg / L for meropenem, between 0.05 and 12 mg / L for the ertapenem.
• Cloxacillin is an antibiotic of the penicillin class. It is used in vitro to inhibit certain beta-lactamases (Giske et al., 2010, supra). Dicloxacillin and flucloxacillin are advantageously considered equivalent to cloxacillin. Preferably, cloxacillin is used at a concentration of between 25 and 300 mg / l.
• PABN or PAbetaN corresponds to phenylalanine-arginine-beta-naphthylamide.
• This compound is known as an inhibitor of efflux pumps, making it possible, for example, to reduce the minimum inhibitory concentration (MIC) of chloramphenicol with respect to strains of Enterobacter aerogenes (Mallea et al., 2002, Biochemical and Biophysical Research Communications 293). : 1370-3).
• MIC minimum inhibitory concentration
• PAbetaN is used at a concentration of between 1 and 50 mg / l.
• chromogenic substrate a substrate allowing the detection of an enzymatic or metabolic activity of the target microorganisms by means of a detectable signal directly or indirectly.
• this substrate may be bound to a fluorescent or colored marker moiety (Orenga et al., 2009, J. Microbiol Methods, 79 (2): 139-55).
• the reaction medium according to the invention may additionally comprise a pH indicator, sensitive to the pH variation induced by the consumption of the substrate and revealing the metabolism of the target microorganisms.
• the said pH indicator may be a chromophore or a fluorophore.
• chromophores examples include bromocresol purple, bromothymol blue, neutral red, aniline blue, bromocresol blue.
• the fluorophores include, for example, 4-methylumbelliferone, hydroxycoumarin derivatives or resorufin derivatives.
• the chromogenic substrate is preferably chosen from substrates based on Indoxyl (3-lndoxyl, 5-Bromo-3-indoxyl, 5-lodo-3-indoxyl,
• the enzymatic activities targeted by the chromogenic substrates may belong to the group of hydrolases, preferentially to the groups of osidases, esterases or peptidases.
• the enzymatic activities targeted by the chromogenic substrates are chosen from: glucuronidase, glucosidase, galactosidase, esterase, sulphatase and deaminase.
• the substrates used for the detection of beta- glucuronidase activity may in particular be 4-methylumbelliferyl-beta-glucuronide,
• the substrates used for the detection of a beta-galactosidase activity may especially be 4-methylumbelliferyl-beta-galactoside, 5-Bromo-4-chloro-3-indolyl-beta-galactoside or 5-Bromo-6-chloro. -3-indolyl-beta-galactoside, the
• the substrates used for the detection of a beta-glucosidase activity may especially be 4-methylumbelliferyl-beta-glucoside, 5-Bromo-4-chloro-3-indolyl-beta-glucoside or 5-Bromo-4-chloro.
• 3-indolyl-N-methyl-beta-glucoside 5-Bromo-6-chloro-3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside, Alizarin-beta-glucoside, Cyclohexenoesculetin-beta-glucoside, Nitrophenyl-beta-glucoside, Dichloroaminophenyl-glucoside or their salts.
• the substrates used for the detection of an esterase activity may in particular be saturated or unsaturated linear fatty acid esters, having between 6 and 14 carbons, preferably between 7 and 9 carbons, and
• 4- Methylumbelliferone, 5-Bromo-4-chloro-3-indoxyl, 5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indolyl or Alizarin or their salts Preferably, they are chosen from 4-methylumbellifl-octanoate, 5-bromo-4-chloro-3-indoxyl-octanoate, 5-bromo-6-chloro-3-indoxyl-octanoate, 6-chloro-3-indoxyl-octanoate, 5-Bromo-3-indolyloctanoate or Alizarin-octanoate.
• the substrates used for the detection of a deaminase activity may in particular be L-Tryptophan, L-Phenylalanine, L-Tyrosine and L-Histidine.
• the substrates used for the detection of a sulphatase activity may especially be 4-methylumbelliferyl sulphate, 5-bromo-4-chloro-3-indoxyl sulphate,
• the chromogenic substrate is chosen from: 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside (X-glucoside), 5-Bromo-6-chloro-3-indoxyl-beta-D- Galactopyranoside (Magenta beta-Gal), 6-Chloro-3-indoxyl-beta-D-glucuronide (Rose beta Gur), 5-Bromo-4-chloro-3-indoxyl-N-methyl-beta-D-glucopyranoside (Green A beta Glu), Methyl-beta-D-glucopyranoside (methyl beta D glucoside), L-Tryptophan.
• Incubate is meant to carry and maintain between 1 and 48 hours, preferably between 4 and 24 hours, more preferably between 16 and 24 hours, at an appropriate temperature, generally between 20 and 50 ° C, preferably between 30 and 40 ° C .
• detection it is meant to detect with the naked eye or with the aid of an optical device the existence of a growth of the target bacteria.
• the detection may also allow identification of the target bacteria. The detection is done with an optical device for fluorescent substrates, or with the naked eye or with an optical device for colored substrates.
• a more specific identification corresponds to a reduction in the number of false positives linked to strains that do not express carbapenemase, without claiming to inhibit all of these strains.
• sensitivity is meant the ability to give a positive result when the target bacterial strain is present in the sample.
• carbapenem enzymatic resistance is meant, as indicated supra, the carbapenem antibiotic resistance due to the expression of carbapenemases by the target bacteria.
• carbapenem-resistant bacteria are, as indicated supra: Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter sp., Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Providencia rettgeri, Pseudomonas putida, Stenotrophomonas maltophilia, Acinetobacter baumanii , Comamonas sp., Aeromonas sp., Morganella morganii, Enterococcus sp., Proteus mirabilis, Salmonella senftenberg, Serratia marcescens, Salmonella typhimurium etc.
• the present invention also relates to a culture medium for detecting and / or identifying bacteria having an enzymatic resistance to carbapenems, said culture medium corresponding to a basic culture medium, further comprising at least one chromogenic substrate, at least one carbapenem, cloxacillin or a combination of cloxacillin and PAbetaN.
• the present invention relates to the use of cloxacillin or a combination of cloxacillin and PAbetaN for specifically detecting and / or identifying bacteria having carbapenem enzyme resistance.
• MIC Minimal Inhibitory Concentrations
• Test B100904 the 19 strains resistant to impermeability (RI) belong to the following species: Enterobacter aerogenes, 2 Enterobacter cloacae, 1 Citrobacter freundii, 1 Pseudomonas aeruginosa, 3 Proteus mirabilis and 2 Morganella morganii.
• Test B101001 Twenty-nine carbapenemase-producing strains are distributed as follows: 27 KPC strains KPC (KPC) and 2 strains respectively Klebsiella pneumoniae and Citrobacter freundii producing class B carbapenemase (type NDM) -1). This test also included an Escherichia coli ESBL strain.
• PabetaN on MICs of RI or carbapenemase-producing strains (Petri dish 90 mm).
• Table III Impact of cloxacillin and / or PAbetaN on the MICs of the carbapenemase-producing strains (comparison with the MICs of the medium without cloxacillin and PAbetaN).
• cloxacillin in the Mueller Hinton medium makes it possible to very clearly reduce (by more than 2 no concentration) the MICs to ertapenem of the majority of the RI strains tested (16/19).
• the addition of PAbetaN alone has no impact on the majority of the RI strains tested.
• the addition of PAbetaN to a medium containing cloxacillin reinforces the effect of the latter by further decreasing the MICs of the RI strains significantly (by more than 2 no concentration for 7 strains) or more moderate (2 no concentration or less for 5 strains).
• Cloxacillin has no impact (9 strains) on MICs on ertapenem-producing strains of carbapenemases (NDM or KPC) or tends to reduce them slightly (16 strains).
• NDM or KPC carbapenemases
• PAbetaN alone in the Mueller Hinton medium tends to increase slightly (15 strains) or very clearly (7 strains) MICs to ertapenem of these strains.
• the combination of cloxacillin and PAbetaN allows results similar to PAbetaN alone, although the positive effect of PAbetaN on MICs is slightly decreased by the negative effect of cloxacillin.
• cloxacillin alone (200 mg / l) makes it possible to significantly reduce the MICs to ertapenem of the majority of the R1 strains tested.
• PAbetaN reinforces this effect of cloxacillin.
• cloxacillin + PAbetaN allows an increase (moderate to high) of the MICs to ertapenem of the carbapenemase producing strains.
• cloxacillin alone or in combination with PabetaN makes it possible to better inhibit the undesirable growth of the IRs, while having no effect, or a slightly positive effect on the growth of the carbapenemase producing strains.
• Class B carbapenemase other than NDM (Class B) 6
• the chromogenic media are divided into 120x120 square boxes.
• Seeding is carried out from 24 h pre-cultures at 37 ° C. on trypcase soya agar. For each strain, a 0.5 McF suspension in physiological saline is performed. Each suspension is inoculated spot (1 to 2 ⁇ ) on each medium using a multipoint inoculator according to the agar dilution method (DEG). Readings are taken after 24 hours of incubation at 37 ° C.
• DEG agar dilution method
• An absence of bacterial growth or a number of colonies of less than or equal to 3 are considered as corresponding to a growth inhibition.
• the presence of 4 or more colonies is considered posiive growth.
• Table VI impact of cloxacillin and / or PAbetaN on the growth (number of developing strains) of the carbapenemase or beta-lactam resistant strains due to another resistance mechanism, in the presence of faropenem T 1 5 9 2 6 10 3 7 1 1 4 8 12 13 16 19 14 17 20 15 18 2
• Table VII Sensitivity and specificity of media containing faropenem in the presence of cloxacillin, combined or with PAbetaN, vis-à-vis carbapenemase-producing or beta-lactam-resistant strains due to another resistance mechanism.
• Table VI II effects of cloxacillin, with or without PAbetaN, on the growth of the 19 R1 strains tested. mg / L) 0 25
• Inhibition of RI strains is observed as soon as 50 mg / L of cloxacillin is added, even more marked for the highest concentrations of faropenem tested (16 and 32 mg / L).
• This inhibitory effect increases with the concentration of cloxacillin (for example, for a concentration of 16 mg / L of faropenem, the addition of cloxacillin at 50 mg / L or 200 mg / L respectively allows the inhibition of 6 and 9 strains of Additional IRs).
• a combined effect of the concentration of faropenem and that of cloxacillin on the RI strains is observed.

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