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Title:
SELECTIVE CULTURE MEDIUM FOR POLYMYXIN-RESISTANT, GRAM-NEGATIVE BACTERIA
Document Type and Number:
WIPO Patent Application WO/2016/151092
Kind Code:
A1
Abstract:
The present invention concerns culture medium for specifically selecting gram-negative, aerobe bacteria that have an acquired resistance to polymyxins as well as bacteria that are naturally resistant to polymyxins. In particular, the culture medium allows selection of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii having an acquired resistance to polymyxins. Presently existing culture media select naturally resistant bacteria, but were found not to select bacteria that have an acquired resistance to polymyxins. The use of polymyxin antibiotics in humans has increased recently, as a last resort treatment against infections by multidrug resistant (MDR) bacteria. The present invention identifies and resolves the need for a diagnostic test for the identification and characterization of bacteria that have an acquired resistance to polymyxins as well.

Inventors:
NORDMANN PATRICE (CH)
POIREL LAURENT (CH)
JAYOL AURÉLIE (CH)
Application Number:
PCT/EP2016/056566
Publication Date:
September 29, 2016
Filing Date:
March 24, 2016
Export Citation:
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Assignee:
UNIV DE FRIBOURG (CH)
International Classes:
C12N1/20; C12R1/01
Domestic Patent References:
WO2008017734A12008-02-14
Other References:
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Attorney, Agent or Firm:
SCHNEITER, Sorin (CH)
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Claims:
A culture medium comprising a polymyxin antibiotic, a daptomycin antibiotic and an antifungal agent.

The culture medium of claim 1, wherein said polymyxin antibiotic is selected from colistin, polymyxin B and mixtures thereof.

The culture medium of claim 1 or claim 2, wherein said polymyxin antibiotic is present at a concentration of 0.01 to 6 μg/ml.

The culture medium of any one of the preceding claims, wherein said polymyxin antibiotic is present at a concentration of 0.4 to 5 μg/ml, preferably 1 to 4 μg/ml, more preferably from > 2 to < 4 μg/ml.

The culture medium of any one of the preceding claims, wherein said daptomycin antibiotic is selected from daptomycin and analogues thereof, preferably it is daptomycin.

The culture medium of any one of the preceding claims, wherein said antifungal agent is amphotericin B.

The culture medium of any one of the preceding claims, wherein the concentration of said daptomycin antibiotic is 0.5 to 50 μg/ml, preferably 1 to 30 μg/ml, more preferably 5 to 15 μg/ml.

The culture medium of any one of the preceding claims, wherein the concentration of said antifungal agent is 0.1 to 50 μg/ml, preferably 0.5 to 25 μg/ml, more preferably 1 to 10 μg/ml.

The culture medium of any one of the preceding claims, further comprising eosine and/or methylene blue, preferably EMB agar.

10. A method for selecting gram-negative, aerobic bacteria that have an acquired resistance to polymyxins as well as bacteria that are naturally resistant to polymyxins, the method comprising the steps of:

providing a culture medium as defined in any one of claims 1-9;

inoculating said culture medium with a sample comprising bacteria;

incubating said inoculated culture medium under conditions suitable for the growth bacteria exhibiting natural or acquired resistance to polymyxins; detecting colonies formed on said inoculated and cultivated medium, said colonies containing said bacteria that exhibit natural and/or acquired resistance to polymyxins.

11. The method of claim 10, wherein said bacteria that have an acquired resistance to polymyxins are bacteria belonging to taxa selected from the group of

Enter obacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii.

12. The method of claim 10, wherein said bacteria that have a natural resistance to polymyxins are bacteria belonging to taxa selected from the group of Burkholderia, Proteus and Serratia.

13. The method of any one of claims 10-12, wherein said incubation step is conducted for 24 hours to 48 hours, preferably 24 to 36 hours.

14. The method of any one of claims 10-13, wherein the detection of a bacteria that have an acquired resistance to polymyxins as well as bacteria that are naturally resistant to polymyxins, with the exception of Burkholderia cepacia and Stenotrophomonas maltophilia, is determined by detecting the occurrence of colonies within a culturing time of 24 to 36 hours.

15. The method of any one of claims 10-14, wherein the detection of naturally resistant Burkholderia cepacia and Stenotrophomonas maltophilia is determined by detecting the occurrence of colonies of a culturing time of 36 hours or more.

Description:
Selective Culture Medium for Polymyxin-Resistant, Gram-Negative Bacteria Technical Field The present invention relates to a culture medium and to a method for specifically selecting gram-negative, aerobic bacteria that have an acquired resistance to polymyxins as well as bacteria that are naturally resistant to polymyxins.

Background Art and Problems Solved by the Invention

Multidrug resistance (MDR) to antibiotics in bacteria is becoming a major issue worldwide. Those bacteria account for most hospital infections worldwide, affecting, for example in the USA, two million people and causing 25,000 deaths annually. Many important public bodies have already raised an alarm signal on the rapid and uncontrollable spread of MDR bacteria such as the WHO, the European Center for Control Diseases in Stockholm and the Center for Diseases control (CDC) in Atlanta (USA), and most recently the International Monetary Fund and the White House (USA).

The most important sources of concern are MDR Gram negative bacteria such as Escherichia coli and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii since those bacterial species are the main sources of infections in humans, with respect to both, community- and hospital-acquired infections. Those infections are mostly urinary tract infections, bloodstream infections, pulmonary and intra-abdominal infections. Their treatment mostly relies on β-lactams such as penicillins, cephalosporins, the broadest spectrum β- lactams i.e. carbapenems, aminoglycosides and quinolones. Acquired resistance to those antibiotic molecules is increasingly reported worldwide, being often associated in MDR bacteria. Very few, if any, therapeutic options are left. Therefore, a renewed interest in an old class of antibiotics, polymyxins, colistin and polymyxin B, is observed worldwide. As a recent example, large use of polymyxins is now observed for treating bloodstream infections due to Klebsiella pneumoniae in Italy which shows a very high prevalence rate (40-60%) of carbapenem resistance due to the spread of carbapenemase producers. The large use of polymyxins in this country explains why colistin resistance superimposes to carbapenem resistance in Enter obacteriaceae.

The polymyxins became clinically available in the 1950's but soon fell out of favor mainly because of concerns of their potential toxic effects for kidneys and development of many efficient and much less toxic antibiotics, in particular cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. Polymyxin molecules were mostly used in veterinary medicine. They have been used in human medicine as an attempt to decolonize patients carrying MDR bacteria. Being products of bacterial fermentation (from Bacillus polymyxa subspecies colistinus), colistin and polymyxin B are multicomponent antibiotics active only against several gram- negative aerobes. They have very similar chemical structures, differing by one amino acid in the peptide ring. The polymyxins are relatively large lipopeptide molecules and are poorly absorbed after oral administration. For the treatment of life-threatening systemic infections, they are given by the intravenous route or by nebulization for the treatment of respiratory tract infections. Likewise other antimicrobial peptides, colistin interacts with the lipid A moiety of the lipopolysaccharide (LPS) of gram-negatives. The polycationic peptide ring competes for and substitutes the calcium and magnesium bridges stabilizing the LPS, promoting membrane permeability and thus disrupting the integrity of the outer membrane of gram negative, thus leading to bacterial death.

Natural polymyxin resistance is known in several gram negatives species such as in Proteus sp, Serratia sp and Burkholderia sp. Acquired resistance to polymyxins is increasingly observed and this is the most important source of concern in the polymyxin resistance domain. Although polymyxin resistance rates remain relatively low in developed countries, except in Italy, Spain and Greece, there is a concern that this situation might change rapidly. The main mechanisms of acquired resistance to polymyxins in Enterobacteriaceae, in particular in K. pneumoniae, are modifications in genes modifying the LPS leading to a weak, if any, binding of polymyxins to the outer-membrane proteins.

Until very recently, acquired resistance to colistin was reported to be based on chromosomal mutations. Therefore, the risk of interspecies transferability of the resistance to colistin was expected to be very low. Very recently, however, Liu et al reported the emergence of plasmid- mediated colistin resistance in China (Liu et al, the list of references is provided at the end of the document). Polymyxin resistance was shown to be due to the plasmid-mediated mcr-1 gene. In a retrospective study, the authors analyzed a collection of E. coli from pigs at slaughter, and found that the presence of mcr-1 increased from year to year. Furthermore, mcr-1 -positive Enterobacteriaceae were also found in the environment and in human inpatients.

Meanwhile, the mcr-1 plasmidic resistance gene was also found in Europe. For example, Nordmann et al (2016) report bacteremia associated with E. coli isolates producing mcr-1 from two patients hospitalized in different hospitals in Switzerland.

Transfer of mcr-1 to carbapenemase producers in nosocomial settings may concretize the apocalypse of antibiotics. Actually, a community-acquired E. coli strain producing mcr-1 and the carbapenemase VIM-1, an E. coli strain co-expressing mcr-1 and kpc-2, and a Klebsiella pneumoniae isolate co-producing mcr-1 and ndm-5 have already been identified. Although The mcr-1 determinant seems so far mostly located in animal and not in human isolates, it brings about further challenges. In particular, the extent to which the mcr-1 gene has disseminated among Enterobacteriaceae needs to be determined. These challenges are complicated by the fact that mcr-1 confers a low level of resistance to polymyxins in E. coli and the common techniques for determining polymyxins susceptibility in routine laboratories are not reliable. Such difficult detection may explain why plasmid-mediated colistin resistance has remained silent until recently, although we now know it has been circulating for at least 10 years.

No medium is available for screening bacteria with acquired resistance to polymyxins that account for variable levels of resistance to polymyxins. Therefore, a selective culture media for screening those polymyxin-resistant strains is needed. Such a medium will be also needed in veterinary medicine to screen for polymyxin-resistant isolates since polymyxins are widely used for prophylaxis, metaphylaxis and treatment of animals, for example, for the treatment of diarrhea in cows. Polymyxins have been used as selective agent as a component of several selective culture media to eliminate polymyxin- susceptible gram negatives from contaminated flora. One of those examples of the use of polymyxin B is the Bacillus anthracis medium. Media containing polymyxins are available for screening bacterial strains that are naturally resistant to polymyxins, such as Burkholderia cepacia and Serratia marcescens. Those media contain high concentrations of colistin and are not adapted for screening isolates with acquired polymyxins resistance, which may be of low-level. Furthermore, such media often contain deoxycholic acids that may interfere with the growth of polymyxins resistant bacteria.

The present invention seeks to address the problems related to multidrug resistance by providing a rapid and innovative test for identifying and characterizing antibiotic-resistant bacteria, in particular bacteria that exhibit an acquired resistance to polymyxins. One goal of the invention is to develop a selective culture medium for screening any type of polymyxin-resistant gram negatives.

Summary of the Invention Remarkably, the inventors propose a method and a medium for detecting bacteria displaying a natural as well as acquired phenotype of resistance to polymyxins.

In an aspect, the present invention provides a culture medium comprising a polymyxin antibiotic, an antibiotic having activity towards gram-positive bacteria, and an antifungal agent.

In an aspect, the present invention provides a culture medium comprising a polymyxin antibiotic, a daptomycin antibiotic and an antifungal agent. In another aspect, the invention provides a method for selecting gram-negative, aerobic bacteria that have an acquired resistance to polymyxins as well as bacteria that are naturally resistant to polymyxins. The method comprising the steps of: providing the culture medium of the present invention; inoculating said culture medium with a sample comprising bacteria; incubating said inoculated culture medium under conditions suitable for the growth of bacteria, in particular bacteria exhibiting natural or acquired resistance to polymyxins; detecting colonies formed on said inoculated and cultivated medium, wherein said colonies containing said bacteria that exhibit natural and/or acquired resistance to polymyxins.

In an aspect, the present invention provides a method for detecting bacteria that have an acquired resistance to polymyxins as well as bacteria that are naturally resistant to polymyxins in a test sample, the method comprising the steps of: providing the culture medium of the present invention; inoculating said culture medium with said test sample; incubating said inoculated culture medium under conditions suitable for the growth of bacteria, in particular of bacteria exhibiting natural or acquired resistance to polymyxins; detecting colonies formed on said inoculated and cultivated medium, said colonies containing said bacteria that exhibit natural and/or acquired resistance to polymyxins.

Further aspects and preferred embodiments are defined in the appended claims and the detailed description herein below.

Brief Description of the Figures

Figure 1 shows polymyxin-resistant lactose-positive E. coli (A), polymyxin-resistant K. pneumoniae (B), polymyxin-resistant lactose-negative E. coli (C), and mix of a heavy inoculum of P. mirabilis and a low inoculum of polymyxin-resistant K. pneumoniae (D) growing on the SuperPolymyxin medium according to an embodiment of the invention.

Detailed Description of the Preferred Embodiments

In an aspect, the present invention provides a culture medium comprising a polymyxin antibiotic, a daptomycin antibiotic and an antifungal agent.

The term "comprising", for the purpose of the present specification, is intended to mean "comprises, amongst other". It is not intended to mean "consists only of . The culture medium of the invention is preferably a gel or liquid designed to support the growth of microorganisms, in particular the growth of gram-negative bacteria. The culture medium may be a nutrient broth or an agar plate, for example. Preferably the culture medium is solid, that is, gel-based. Preferably, the culture medium is agar-based. The culture medium of the invention is preferably a selective medium, supporting the growth of selected microorganisms only. In an embodiment, the culture medium is a selective as well as an indicator medium, which allows identifying characteristics of the microorganism growing on the medium.

The culture medium preferably comprises nutrients for supporting the growth of the microorganisms that are to be selected. The culture medium preferably comprises a carbon source, in particular carbohydrates, such as poly-, oligo-, di- and/or monosaccharides that can be metabolized by the bacteria to be selected. Preferably, the culture medium comprises di- and/or monosaccharides. In an embodiment, the culture medium may contain one or more selected from sucrose, glucose and lactose. In a preferred embodiment, the culture medium contains lactose and sucrose as carbon sources supporting the growth of microorganisms. As a source of amino acids, the culture medium may contain peptone, for example. The culture medium preferably comprises various salts needed for bacterial growth.

In a preferred embodiment, the culture medium of the invention is based on and/or comprises Eosine Methylene Blue (EMB) Culture Medium, according to the formulation described by M. Levine in 1918 J Infect Dis 23: 43-47). The EMB medium is a selective-differential plating medium for the detection and isolation of gram-negative bacteria.

The present invention seeks to provide a culture medium that allows for the rapid detection of bacteria that have an acquired resistance as well as bacteria that have a natural resistance to polymyxins antibiotics. Therefore, the culture medium comprises a polymyxin antibiotic. In an embodiment, the polymyxin antibiotic is selected from colistin, polymyxin B and mixtures thereof. In an embodiment, the culture medium comprises only one polymyxin antibiotic, which is colistin. Preferably, colistin is colistin according to CAS number 1264-72-8.

In another embodiment, the culture medium comprises only one polymyxin antibiotic, which is polymyxin B. Preferably, polymyxin B is the molecule according to CAS number 1405-20- 5.

In an embodiment, the said polymyxin antibiotic is present at a concentration of 0.01 to 6 μg/ml in said culture medium. Preferably, this concentration applies to the total concentration of all polymyxins present in the culture medium, in case a mixture of different polymyxin antibiotics, such as a mixture of colistin and polymyxin B is present in the culture medium.

In a preferred embodiment, said polymyxin antibiotic is present at a concentration of 0.4 to 5 μg/ml, preferably 1 to 4 μg/ml, more preferably from > 2 to < 4 μg/ml.

In a most preferred embodiment, said polymyxin antibiotic is present at a concentration of 3.0 to 4 μg/ml, for example 3 to 3.6 μg/ml. The inventors found that for identifying bacteria that have resistance to polymyxin antibiotics, concentrations more than 2 μg/ml are preferred. This is surprising, given that the EUCAST (European Committee in Antimicrobial Susceptibility Testing) has determined the minimum inhibitory concentration (MIC) values of polymyxins to be < 2 mg/L and resistance >2 mg/L for Enter obacteriaceae and Acinetobacter baumannii, and susceptibility < 4 mg/L and resistance >4 mg/L for Pseudomonas aeruginosa. The present inventors have identified a polymyxin concentration that is suitable to select for most if not all types of resistances, including acquired resistances, which are generally not susceptible but not resistant to polymyxins, either. The medium of the present invention is suitable to select for bacteria that are "naturally resistant" to polymyxins. This group of bacteria contains bacteria that have originally never been susceptible to polymyxins, in particular in the taxa of Proteus sp, Serratia sp and Burkholderia sp.

On the other hand, the medium of the present invention is intended to select for, detect and/or identify bacteria that have an acquired resistance to polymyxins. This group of bacteria has developed a resistance or a partial resistance upon exposure to the antibiotic and is thus different from the naturally resistant group. Acquired resistance is observed in other bacterial taxa, in particular in Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae; Acinetobacter baumannii, and Pseudomonas aeruginosa. Table 2 below provides a list containing bacterial taxa that are reported to have acquired partial or total resistance.

Surprisingly, the medium of the present invention is also suitable to detect and select for bacteria, in particular Enterobacteriaceae, which have the acquired resistance to polymyxins mediated by the mcr-1 gene. The mcr-1 mediated resistance is the first ever reported resistance to polymyxins mediated by a plasmid and being thus transferable between bacterial species. Advantageously, the invention provides a method for selecting for and/or detecting gram-negative, aerobic bacteria that have an acquired resistance to polymyxins, including bacteria that carry the plasmid containing mcr-1.

The present inventors observed that bacteria having an acquired resistance to polymyxins are generally not as resistant to polymyxins as the naturally resistant bacteria. Bacteria having an acquired resistance to polymyxins are generally partially resistant and/or partially susceptible to polymyxins. Furthermore, the resistance and/or susceptibility of bacteria having an acquired resistance are highly variable and based on various different molecular mechanisms.

For this reason it is surprising to provide a selective medium containing polymyxins at a determined concentration or concentration range that is suitable to select for all bacteria having an acquired resistance to polymyxins, or at least all bacteria that were tested in the experiments conducted by the inventors.

In a preferred embodiment, the culture medium of the invention comprises an additional antibiotic active against gram-positive bacteria. Preferably, the additional antibiotic is daptomycin. In a preferred embodiment, the culture medium of the invention comprises a daptomycin antibiotic. The daptomycin antibiotic is preferably selected from daptomycin and analogues thereof, preferably it is daptomycin. In a preferred embodiment, daptomycin is daptomycin having CAS number 103060-53-3.

The present inventors observed that, if an EMB culture medium is used, the growth of gram- positive bacteria is not sufficiently inhibited by the dye methylene blue and eosin. Therefore, the inventors determined that a further antibiotic, specific to gram-positive bacteria, is preferably present. The present inventors have tested several antibiotics to this end, but not all of the tested antibiotics fulfilled the purpose in accordance with the present invention. Surprisingly, excellent results were achieved when using daptomycin, in particular in connection with said EMB medium.

In an embodiment, the additional antibiotic active against gram-positive bacteria comprises daptomycin and is substantially free of vancomycin, since vancomycin potentiates the activity of colistin against several gram negatives. "Substantially free", for the purpose of this specification, means less than 5 mol.%, preferably less than 2 mol.%, more preferably less than 1 mol.% and most preferably less than 0.5 mol.% with respect to the total of antibiotics including said polymyxins antibiotic, but excluding said antifungal agent. Preferably, vancomycin is entirely absent in the medium of the invention.

In an embodiment, the concentration of said daptomycin antibiotic in the culture medium of the invention is 0.5 to 50 μg/ml, preferably 1 to 30 μg/ml, more preferably 5 to 15 μg/ml. A concentration of 8 to 12 μg/ml is most preferred.

The culture medium of the present invention preferably comprises an antifungal agent. Preferably, the antifungal agent is amphotericin B.

In an embodiment, the concentration of said antifungal agent, in particular amphotericin B in the culture medium of the invention is 0.1 to 50 μg/ml, preferably 0.5 to 25 μg/ml, more preferably 1 to 10 μg/ml, and most preferably 3-7 μg/ml. In an embodiment, the culture medium of the invention is EMB agar which contains peptone, lactose, sucrose, and the dyes eosin and methylene blue. It is commonly used as both a selective medium for Gram-negative bacteria and a differential medium. As mentioned above, the combination of eosin and methylene blue dyes inhibits most gram- positive bacteria and allows the selection of the gram-negative bacteria. The eosin dye allows also the differentiation between lactose fermenters and lactose non fermenters. It responds to decrease in pH when lactose is fermented, going from colorless to black. Therefore, lactose fermenters as E. coli and E. cloacae possess dark centers when cultured on EMB a medium containing eosin and methylene.

On the other hand, sucrose included in an eosin and methylene blue-containing medium allows the differentiation between coliforms that are able to ferment sucrose more rapidly than lactose and those that are unable to ferment sucrose. The vigorous lactose and sucrose fermentation of E. coli on a medium containing lactose and sucrose as a carbon sources causes a bigger amount of acid production than for only lactose-fermenting bacteria and is associated with a green metallic sheen. Therefore, E. coli bacteria will give a distinctive green-metallic sheen with a dark center. Lactose or sucrose non-fermenters as Proteus mirabilis are colorless and appear therefore translucent or pink.

In an embodiment, the culture medium comprises chromogenic components, in particular chromogenic components other than and/or in addition to eosine and methylene blue. Such chromogenic components may allow the rapid recognition of bacterial taxa. In a preferred embodiment, the culture medium comprises chromogenic compounds for specifically detecting polymyxin resistant Acinetobacter baumannii and Pseudomonas aeruginosa. For example, the presence of β-alanylarylamidase activity in certain bacteria, such as Pseudomonas, can be determined using 7-Amido-l-pentyl-phenoxazin-3-one or other chromogenic compounds in the culture medium. In a preferred embodiment, the present invention provides an EMB agar-based culture medium comprising a polymyxin selected from colistin and polymyxin B, daptomycin and amphotericin B. In a preferred embodiment, this culture medium comprises 1-5 μg/ml of said polymyxin; 2 to 20 μ§/ι 1 of daptomycin and 1-10μ§/ιη1 of amphotericin B.

In a more preferred embodiment, the invention provides an EMB agar gel-based culture medium comprising more than 2 and less of 4 μg/ml of said polymyxin, 5 to 15 μg/ml of daptomycin and 3-9 μg/ml of amphotericin B.

In a most preferred embodiment, the invention provides an EMB agar gel-based culture medium comprising 3 to 3.6 μg/ml of said polymyxin, 5 to 15 μg/ml of daptomycin and 3- 9 μg/ml of amphotericin B.

The present invention provides a method for identifying bacteria having an acquired or a natural resistance to polymyxins and a method for selecting and/or detecting such bacteria.

The methods of the invention preferably comprise the steps of:

- providing a culture medium of the present invention;

inoculating said culture medium with a test sample;

incubating said inoculated culture medium under conditions suitable for the growth of bacteria, in particular for the growth of bacteria exhibiting natural or acquired resistance to polymyxins;

- detecting colonies formed on said inoculated and cultivated medium, said colonies containing said bacteria that exhibit natural and/or acquired resistance to polymyxins.

For the purpose of the present specification, a "test sample" means any liquid or solid material to be tested, which may contain bacteria that have an acquired or natural resistance to polymyxins. Preferably, the "test sample" is a biological sample.

For the purpose of the present specification, a "biological sample" means any biological sample derived from a subject. Examples of such samples include body fluids, skin swabs, tissues, cell samples, etc. Preferred biological samples are saliva, whole blood, serum, plasma, urine or faeces.

In an embodiment, the method of the invention can be applied directly to a raw test sample, such as a raw biological sample, comprising a mixture of bacteria, without having to isolate or separate the various bacterial strains present in the sample.

The culture medium and the methods of the invention have the advantage of eliminating polymyxin-susceptible gram negatives, gram-positives and fungi that are usually part of many bacterial flora.

As used herein, a "subject" denotes a human or animal subject, wherein said animal subject is preferably a mammal. The animal subject may be selected from artyodacyla, in particular land- living, domesticated artyodactyla, such as cattle and pigs, perissodactyla, such as horses, carnivores, such as dogs and cats, rodents and primates.

In a preferred embodiment, the "subject" is a human or a domestic animal, in particular a human or a livestock animal or companion animals

The methods of the invention may be used to detect bacteria that exhibit at least a partial resistance to polymyxins, such as bacteria having a reduced susceptibility, which means a decreased susceptibility compared to wild-type polymyxin susceptibility. In an embodiment of the methods and the culture medium of the invention, said bacteria that have an acquired resistance to polymyxins are bacteria belonging to taxa selected from the group of Enter obacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii.

In an embodiment of the methods and the culture medium of the invention, said bacteria that have a natural resistance to polymyxins are bacteria belonging to taxa selected from the group of Burkholderia, Proteus and Serratia.

In an embodiment of the methods of the invention, said step of providing a culture medium in accordance with the invention comprises mixing the components of the culture medium and thereby obtaining said culture medium. Active components of the culture medium may be provided from stock solutions, which may be added or mixed with the solution of the medium to be prepared, such as a solution of agar in water, for example. Active components are in particular said polymyxin antibiotic, said daptomycin antibiotic and said antifungal agent. Preferably, the stock solution of said polymyxins antibiotic, in particular if the polymyxin is colistin, is prepared with the use of glass tubes and/or is provided in a glass recipient, to avoid the binding of the colistin to polystyrene, for example. Alternatively, said tubes or recipient may be made from a plastic polymer other than polystyrene or from a plastic polymer to which colistin does not stick. The use of an appropriate material may allow avoiding the use of a surfactant, such as Tween80 in a solution containing polymyxins. In other embodiments, the present invention does not exclude the presence of a surfactant, such as tween, e.g. tween80, in the medium of the invention.

In an embodiment of the methods of the invention, said step of incubating said inoculated culture medium under conditions suitable for the growth of bacteria exhibiting natural or acquired resistance to polymyxins (said incubation step), is conducted for 24 hours to 48 hours, preferably 24 to 36 hours.

Conditions suitable for the growth of bacteria exhibiting natural or acquired resistance to polymyxins can be determined by the skilled person. Typically, such conditions include a temperature of about 30-39°C, preferably 35-37°C, the presence of water, salts, and macro and micronutrients required for bacterial growth. Such nutrients are contained, for example, in the EMB agar as disclosed elsewhere in this specification. Other media are likewise suitable to provide nutrients suitable for bacterial growth in accordance with the invention.

In an embodiment of the methods of the invention, the detection of bacteria that have an acquired resistance to polymyxins is determined by detecting the occurrence of colonies within a culturing time of 24 to 36 hours. Bacteria that are naturally resistant to polymyxins, such as Burkholderia cepacia and Stenotrophomonas maltophilia, are also detected within this time frame.

The step of detecting colonies formed on said inoculated and cultivated medium is preferably accomplished by visual inspection. In particular, the presence of colonies is determined visually by a trained person capable of identifying bacterial colonies. The presence of such colonies indicates the presence of growth of bacteria, wherein growth generally means exponential growth. Accordingly, the presence of a colony on the culture medium indicates that said bacteria are in an exponential growth phase within the above specified time frames of 24 hours to 48 hours, preferably 24 to 36 hours. In an embodiment, the detection of naturally resistant Burkholderia cepacia and Stenotrophomonas maltophilia is determined by detecting the occurrence of colonies at a culturing time of 36 hours or more. Surprisingly, these naturally resistant bacteria generally enter an exponential growth only at or after 36 hours on the culture medium of the present invention.

Examples

Material and Methods

Antimicrobial agents, reagents, and materials. Standard colistin sulfate and polymyxins B were from Sigma Aldrich (Saint-Louis, USA). Those antibiotic powders were kept at 4°C before their use and polymyxin dilutions were kept at -20°C. They were used for MIC values determination and for determining the optimal polymyxin concentration to be used in the SuperPolymyxin medium. Daptomycin and amphotericin B were from Novartis (Horsham, UK) and Bristol-Myers-Squibb (Reuil-Malmaison, France), respectively. Cation-adjusted Mueller-Hinton powder from bioRad (Marnes-La-Coquette, France) were used for MIC determinations. Eosine Methylene Blue (EMB) medium was from Fluka, Saint-Louis, USA.

Isolates. A total of 88 isolates were included in the study to evaluate the performance of the SuperPolymyxin medium. The isolates included fifty-two PR (polymyxin-resistant) gram- negatives of worldwide origin and from human and veterinary medicine (Table 2, part I). Seven strains were intrinsic PR genders (Morganella, Proteus, Providencia, Serratia, and Burkholderia). Fourty-five isolates with acquired PR were also included being 36 enterobacterial strains (E. coli, Klebsiella spp., Enterobacter cloacae, and Hafnia alvei) and 9 non-fermenters (Acinetobacter baumannii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia). Thirty-one polymyxin-susceptible strains were included with 23 enterobacterial strains and 8 non-fermenters (Table 2, parts I and II). Determination of MIC values. MICs of polymyxins (colistin and polymyxin B) were determined using the broth microdilution method in cation-adjusted Mueller-Hinton broth, as recommended by the CLSI (Clinical and Laboratory Standards Institute). A final inoculum of 5.10 5 CFU/ml of each strain was distributed in the 96 polystyrene wells tray (Sarstedt, Numbrecht, Germany). E. coli ATCC25922 and P. aeruginosa ATCC27853 were used as polymyxin- susceptible control strains (CLSI 2012 reference, see further below), and all experiments were repeated in triplicate. The results were interpreted according to the CLSI breakpoints (CLSI 2014 reference) for A. baumannii (susceptible [S] < 2 μg/ml, resistant [R] > 4 μg/ml) and for Pseudomonas spp. (S < 2 μg/ml, R > 8 μg/ml) and according to the EUCAST breakpoints for Enterobacteriaceae, i.e. S < 2 μg/ml and R > 2 μg/ml (EUCAST reference). MIC values for colistin and polymyxin B were superimposable, permitting an easy classification between PR and polymyxin- susceptible strains (Table 2, part I).

Determination of the resistance mechanism. The known genes possibly involved in chromosomally-encoded PR in E. coli, K. pneumoniae, K. oxytoca, P. aeruginosa and A. baumannii were sequenced as described previously (Adams et al, Jayol et al 2015, Lee et al, Poirel et al 2014, Quesqdq et al). The PCR detection of the mcr-1 gene was carried out as described in Bontron et al (Table 2, part II). MIC values of polymyxins for strains with acquired resistance were variable and lower than those for intrinsically PR strains (Table 2, parts I and II).

The Super Polymyxin medium. Several colistin and polymyxin B concentrations were studied and colistin at a concentration of 3.5 mg per L was retained to be added to select for colistin resistant strains.

The SuperPolymyxin medium contains Eosine Methylene Blue (agar) as described by Levine in 1918. It is able to select for gram negatives distinguishing between lactose-positive and lactose-negative bacteria. Escherichia coli grow with a green metallic color with a dark center whereas Klebsiella pneumoniae grows with a brownish color with dark-centered mucoid colonies.

Since some Streptococcus and Staphylococcus strains may still grow on EBM medium as small colony variants, daptomycin was added at a concentration of 10 μ§/ι 1. Noteworthy, daptomycin and not vancomycin was added since vancomycin potentiates the activity of colistin against several gram negatives (Gordon et al, Randall et al). The stock solutions of colistin, daptomycin, and amphotericin B were prepared as indicated in Table 1, and may be kept at -20°C during one year. Noteworthy, glass tubes were used to prepare colistin stock solutions to avoid its binding to polystyrene. Colistimethate sulfate, a therapeutic prodrug of colistin, cannot be used. The diluted powder of EMB was autoclaved at 121°C for 15 min. After cooling the EMB medium for one hour at 56°C, the antibiotic stock solutions were added. Poured plates were stored at 4°C and protected from direct light exposure up to one week.

Table 1 : The Preparation of the SuperPolymyxin medium

lume of 400 ml of SuperPolymyxin medium was for i.e. twenty plates.

Screening for polymyxin resistance by using the SUPERPOLYMYXIN medium. Using an inoculum with an optical density of 0.5 McFarland (inoculum of ~10 8 CFU/ml), serial 10-fold dilutions of the isolates were made in normal saline and 100-μ1 portions were plated onto the SuperPolymyxin medium. To quantify the viable bacteria in each dilution, trypticase soy agar was inoculated concomitantly with 100 μΐ of suspension and incubated overnight at 37°C. The number of viable colonies was counted after 24 h of culture at 37°C (and after 36 and 48 h for B. cepacia, P. aeruginosa and S. maltophilia). PR and polymyxin- susceptible control strains were FR-01 (Morganella morganii) and FR-136 (E. coli ATCC25922), respectively (Table 2, parts I and II). The lowest limit of detection for the tested strains were determined using the SuperPolymyxin medium. The sensitivity and specificity cut-off values were set at 1 x 10 3 CFU/ml i.e., a limit value of 1 x 10 3 CFU/ml and above was considered as «not efficiently detected)) (Nordmann et al, 2012). Screening of spiked stool samples. Spiked stools were also tested with a representative collection of 22 PR isolates of various species with various levels and mechanisms of PR (Table 2). Spiked fecal samples were made by adding 100 μΐ of each strain dilution to 900 μΐ of fecal suspension that was obtained by suspending 5 g of freshly pooled feces from five healthy volunteers in 50 ml of distilled water, as described previously (Naas et al). A non- spiked fecal suspension was used as negative control. The lowest detection limit of the PR isolate was determined by plating 100 μΐ of each dilution on the medium. The sensitivity and specificity were determined using a same cut-off value set at > 10 3 CFU/mL (Nordmann et al 2012). This value may correspond to a low-level carriage of PR bacteria in stools.

Table 2 (part I): Bacterial strains tested and determination of MICs of polymyxins

MIC a Polymyxin

Strain Species Origin Colistin Polymyxin B resistance

Yeasts isolates

FR-A C. albicans France NA NA NA

Gram-positive cocci isolates

FR-B S. aureus France NA NA NA

FR-C S. epidermidis France NA NA NA

FR-D E. faecium France NA NA NA

FR-E E. faecalis France NA NA NA

Gram-negative rods isolates naturally resistant to polymyxins

FR-01 M. morganii France >128 >128 R

FR-02 P. mirabilis France >128 >128 R

FR-03 P. vulgaris France >128 >128 R

FR-04 P. stuartii France >128 >128 R

FR-05 S. marcescens France >128 >128 R

FR-201 B. cepacia France >128 >128 R

FR-202 B. gladioli France >128 >128 R

Gram-negative rods isolates with an acquired mechanism of resistance to

polymyxins

FR-06 K. pneumoniae France 32 64 R

FR-07 K. pneumoniae France 32 32 R

FR-09 K. pneumoniae Turkey 32 64 R

FR-10 K. pneumoniae South Africa 16 8 R

FR-17 K. pneumoniae Turkey <128 128 R

FR-21 K. pneumoniae France 32 64 R

FR-30 K. pneumoniae France >128 64 R

FR-31 K. pneumoniae France 64 32 R

FR-36 K. pneumoniae Colombia 128 128 R

FR-40 K. pneumoniae France 64 64 R

FR-41 K. pneumoniae France >128 128 R FR-47 K. pneumoniae Turkey 64 32 R

FR-48 K. pneumoniae Spain 128 128 R

FR-49 K. pneumoniae France 64 32 R

FR-54 K. pneumoniae Colombia 128 128 R

FR-56 K. pneumoniae Spain 64 64 R

FR-68 K. pneumoniae Colombia 64 64 R

FR-70 K. pneumoniae Colombia 128 128 R

FR-71 K. pneumoniae Turkey 32 32 R

FR-86 K. pneumoniae Spain 64 64 R

FR-89 K. pneumoniae Colombia >128 >128 R

FR-92 K. oxytoca Colombia 64 64 R

FR-93 E. coli France 4 4 R

FR-94 E. coli South Africa 16 16 R

FR-98 E. coli South Africa 8 8 R

FR-203 A. baumannii Switzerland 128 128 R

FR-100 K. pneumoniae France 64 64 R

FR-102 K. pneumoniae France 32 32 R

FR-113 K. pneumoniae France >128 >128 R

FR-114 K. pneumoniae Colombia 64 128 R

FR-116 K. pneumoniae France 64 32 R

FR-119 E. coli France 8 4 R

FR-120 E. coli France 8 4 R

FR-121 E. coli France 4 4 R

FR-122 E. cloacae Colombia 32 16 R

FR-126 E. cloacae France >128 >128 R

FR-135 H. alvei France 16 8 R

FR-204 A. baumannii USA >128 >128 R

FR-205 A. baumannii USA 8 8 R

FR-206 A. baumannii USA >128 >128 R

FR-207 P. aeruginosa Colombia 64 64 R

FR-208 P. aeruginosa France >128 >128 R

FR-209 P. aeruginosa France >128 >128 R

FR-210 S. maltophilia France >128 >128 R

FR-211 S. maltophilia France 32 32 R

Gram-negative rods isolates susceptible to polymyxins

FR-136 E. coli ATCC 25922 0.25 0.25 S

FR-212 E. coli USA 0.12 0.12 S

FR-213 E. coli Colombia 0.12 0.12 s

FR-214 E. coli France 0.12 0.12 s

FR-215 E. coli France 0.25 0.12 s

FR-216 E. coli France 0.25 0.12 s

FR-217 K. pneumoniae USA 0.12 0.12 s

FR-218 K. pneumonia Colombia 0.12 0.5 s

FR-219 K. pneumoniae Colombia 0.12 0.12 s

FR-220 K. pneumoniae Colombia 0.5 0.25 s

FR-221 K. pneumoniae Colombia 0.5 0.5 s FR-222 K. pneumoniae France 0.12 0.25 S

FR-223 K. pneumoniae France 0.12 0.12 S

FR-224 K. pneumoniae France 0.25 0.5 S

FR-225 K. pneumoniae France 0.5 0.5 S

FR-226 K. pneumoniae Spain 0.5 0.5 S

FR-227 K. pneumoniae Spain 0.5 0.5 S

FR-228 E. cloacae Colombia 0.12 0.25 S

FR-229 E. cloacae Colombia 0.12 0.12 S

FR-230 E. cloacae France 0.12 0.25 S

FR-231 E. aerogenes France 0.12 0.12 S

FR-232 C. freundii Colombia 0.25 0.25 S

FR-233 C. freundii France 0.12 0.5 S

FR-234 P. aeruginosa ATCC 27853 0.5 0.5 S

FR-235 P. aeruginosa Colombia 1 0.25 S

FR-236 P. aeruginosa Colombia 2 0.5 S

FR-237 P. aeruginosa Colombia 1 0.25 S

FR-238 P. aeruginosa France 2 0.25 S

FR-239 A. baumannii Colombia 0.5 0.12 S

FR-240 A. baumannii France 0.5 0.25 S

FR-241 A. baumannii USA 0.25 0.25 S a MICs of colistin and polymyxin were determined by broth microdilution method.

Table 2 (part II): Limits of detection of the SuperPolymyxin medium.

polymyxins

ram-negatve ro s soates suscept e to poymyxns

b Underlined CFU counts are considered as negative results (cutoff values set at > 1.10 3 CFU/ml)

c Positive culture after 36 hours

d Positive culture after 48

Results

The use of the EMB medium with the added components permits a visual identification of many enterobacterial species according to their color. The medium differentiates lactose fermenters (black colonies) from non-fermenters (colorless or light lavender) (Figure 1). Moreover, differentiation of lactose fermenters was possible with Escherichia coli colonies displaying a characteristic metallic green sheen (Figure 1 A) while Enterobacter spp. and Klebsiella spp. giving brown, dark-centered, and mucoid colonies (Figure 1 B).

A further modification of the screening media could be addition of other chromogenic components for detecting specifically polymyxin-resistant A. baumannii and P. aeruginosa.

All the PR strains grew on the SuperPolymyxm medium in 24 h except P. aeruginosa, S. maltophilia and the intrinsically PR Burkolderia genus that grew in 24 to 48 h (Table 2, part II). The lowest limit of detection was below the cut-off value for all PR strains, whereas the limit of detection of the polymyxin-susceptible strains was above, being > 1 x 10 6 CFU/ml (Table 2, part II). The sensitivity and specificity of the SuperPolymyxm medium for selecting PR isolates were 100% in both cases. Moreover, this medium was tested with a light growth of PR-resistant K. pneumoniae isolate (FR-10) among a heavier growth of Proteus mirabilis (FR-02) and revealed that it nicely discriminated between the two species (Figure 1 D). All the PR isolates spiked in stools grew with a lowest detection limit ranging from 10 1 to 10 2 CFU/ml (Table 2, part II).

Similar results were obtained with 20 colistin/polymyxin B-susceptible strains and 20 colistin/polymyxin B-resistant strains by using polymyxin B at the same concentration instead of colistin sulfate.

Finally, to assess the storage stability of the SuperPolymyxm medium, C. albicans, S. aureus and colistin- susceptible E. coli ATCC 25955 were subcultured daily onto SuperPolymyxm agar plates from a single batch of medium stored at 4°C. Growth of those isolates were consistently inhibited during at least a 7-days period.

The colistin-containing SuperPolymyxm medium was developed for screening polymyxin- resistant gram negatives. It was evaluated with 88 polymyxin-susceptible or -resistant cultured gram-negative isolates. Its sensitivity and specificity of detection were ca. 100%. The SuperPolymyxm medium is the first screening medium able to detect intrinsic and acquired polymyxin-resistant bacteria. The SuperPolymyxin medium constitutes a screening medium aimed to detect any PR bacteria regardless of its resistance mechanism and of its level. This medium may be used in human medicine for detecting carriers and in veterinary medicine for surveillance surveys. References

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