METHOD FOR PREDICTING THE VIRULENCE AND PATHOGENICITY OF GRAM-NEGATIVE BACTERIAL STRAINS

Abstract

A method for predicting the pathogenicity and virulence of a strain of Gram-negative bacteria of the Enterobacteriaceae family, wherein the amount of 2-hydroxymyristate and/or 2-hydroxymyristic acid present in the lipopolysaccharides of the bacteria is identified and measured, the amount of 2-hydroxymyristate and/or 2-hydroxymyristic acid is compared with a reference value, and wherein it is concluded that the strain is virulent if the amount of 2-hydroxymyristate is greater than the reference value. Also, the use of the 2-hydroxymyristic acid ester as a marker of pathogenicity and virulence of a Gram-negative bacterial strain and an in vitro diagnosis kit implementing this marker.

Claims

1.-12. (canceled)

13. A method for predicting the pathogenicity of a strain of Gram-negative bacteria of the Enterobacteriaceae family, wherein the amount of 2-hydroxymyristate and/or 2-hydroxymyristic acid present in the lipopolysaccharide of the bacteria is measured, said amount of 2-hydroxymyristate and/or 2-hydroxymyristic acid is compared with a reference value, and wherein it is concluded that the strain is pathogenic if the amount of 2-hydroxymyristate and/or 2-hydroxymyristic acid is greater than the reference value.

14. The method as claimed in claim 13, wherein the bacterium belongs to the genus Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonella, Serratia or Yersinia.

15. The method as claimed in claim 13, wherein the bacterium belongs to the genus Enterobacter, Escherichia, Pseudomonas, Serratia or Yersinia.

16. The method as claimed in claim 13, wherein the Gram-negative bacteria are bacteria of the genus Enterobacter belonging to the species of the Enterobacter cloacae complex.

17. The method as claimed in claim 13, wherein the amount of 2-hydroxymyristate is compared with the amount of 3-hydroxymyristate present in the lipopolysaccharide of the bacteria, and in that if the ratio of 2-hydroxymyristate to 3-hydroxymyristate is greater than 0.01, it is concluded that the strain is pathogenic.

18. The method as claimed in claim 16, wherein the method is a method for predicting the resistance of strains of Enterobacter cloacae to antibiotics and/or to the responses of the host to the antimicrobial peptides produced by the host.

19. The method as claimed in claim 13, wherein the bacteria are present in a biological sample.

20. The method as claimed in claim 19, wherein the biological sample originates from a mammal, preferably from a human being, and is selected from blood, serum, plasma, cerebrospinal fluid (LCR), ascites, pleural fluid, mucus, stools, or a mucocutaneous sample.

21. The method as claimed in claim 13, further comprising a prior step of isolating the bacteria from a sample.

22. The method as claimed in claim 13, wherein the reference value is equal to 0.

23. A method of predicting the pathogenicity and/or of virulence of a Gram-negative bacterial strain using a 2-hydroxymyristic acid ester as a marker thereof, comprising measuring a concentration of 2-hydroxymyristic acid ester in a Gram-negative bacterial strain selected from bacteria of the genus Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonella, Serratia and/or Yersinia, (in particular of Enterobacter cloacae complex).

24. A kit for diagnosing the pathogenicity of a strain of Enterobacter cloacae complex, comprising means for measuring the concentration of 2-hydroxymyristate in the LPS of a bacterial isolate.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0136] The invention will be better understood from reading the following examples. In these examples, reference will be made to the following figures:

[0137] FIG. 1 Negative-ion MALDI/TOF mass spectrometry of lipid A of the E. cloacae type E strain with cephalosporinase overproduction. P, L-Ara4N, and C16 represent m/z movements corresponding to the phosphate, 4-amino-4-deoxy-L-arabinose, and palmitate substituents, respectively. The marked peaks represent the species where C14:0 is replaced by C12:0 (.diamond-solid.) or 2-hydroxymyristate (•);

[0138] FIG. 2 Negative-ion MALDI/TOF mass spectrometry of the E. cloacae strain lipid A, untreated or hydrolyzed with HCl;

[0139] FIG. 3 Structure of the main molecular species of the E-type lipid A of E. cloacae. Structures A and B are present in almost equal amounts. [x] minor substituents: L-Ara4N/PO3H2; [y] minor variants: C12:0/C13:0/2OH—C14; [z] minor substituent: C16:0;

[0140] FIG. 4 Gas chromatogram of the LPS fatty acid methyl esters of the strain of E. cloacae H7i;

[0141] FIG. 5 Survival analysis according to Kaplan-Meyer as a function of the presence of LPS carrying 2OH—C14. The solid line corresponds to the strains for which 2-hydroxymyristic acid is present in the LPS. The dashed line corresponds to strains for which 2-hydroxymyristic acid is absent in the LPS;

[0142] FIG. 6 Survival test of strains of Enterobacter cloacae complex expressing 2-hydroxymyristate and not expressing 2-hydroxymyristate, after 4 hours of exposure to different concentrations of polymyxin B.

EXAMPLE 1

[0143] 18 patients from the neonatal intensive care unit of the Antoine Béclère hospital, Assistance-Publique Hôpitaux de Paris (Paris Public Health Care Hospitals) (Clamart, France), all very premature (<28 weeks of gestational age), were infected with E. cloacae. The strains were isolated and twelve of them, which exhibited several different characteristics, in particular ERIC-PCR profiles, were selected as shown in Table 1.

TABLE-US-00001 TABLE 1 Susceptibility to 3.sup.rd generation Isolate Source of isolation cephalosporins ERIC-PCR profile H1  Blood R A H2  Blood R A H7o Blood R E H7i Blood S E H8  Blood S C H9  Blood S B H10 Blood S F H11 Blood R A C12 Cavum S H C16 Cavum S G C17 Cavum S E C18 Cavum S F

[0144] To this end, the bacterial isolates were inoculated on a Columbia agar containing 5% of sheep blood ((bioMérieux, Marcy l'Étoile, France) and were incubated overnight at 37° C. Bacterial identification was confirmed by mass spectrometry (MALDI-TOF) (Brucker, Leipzig, Germany).

[0145] Antimicrobial susceptibility testing was performed using the agar disk diffusion method on Mueller-Hinton (MH, Bio-Rad, Hercules, Calif.) according to CLS138.

[0146] Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) was carried out in accordance with Duan et al. (Environ. Res. 109, 511-517; 2009). The ERIC band profiles obtained from agarose gel electrophoresis were used to define different profiles.

[0147] Composition of Molecular Species Present in Lipid A Regions.

[0148] The lipopolysaccharides were extracted from the cultured strains and the structures of their lipid A regions were analyzed using MALDI-TOF.

[0149] The bacteria were grown overnight at 37° C. in LB broth (Sigma) and the LPSs were isolated using the phenol/water method of Westphal and Jann (40). Briefly, the wet pellet of bacteria was shaken in a 45% aqueous phenol solution at 65° C. for 30 min, insoluble matter was removed from the cooled aqueous phase by centrifugation, and the clear extract was dialyzed in tap water until the phenol was eliminated, then dialyzed against distilled water. The extracts were subjected to enzymatic treatments (DNase, RNase and proteinase K) to remove DNA, RNA and proteins, then purified with acidified chloroform-methanol-water to remove contaminating phospholipids and lipoproteins. The LPSs were then washed in suspension in cold methanol, centrifuged (7000×g) and dried under a stream of air. Lipid A was prepared using the triethylamine-citrate method. Briefly, the LPS sample was suspended at a concentration of 10 μg/μl in a 0.01 M triethylamine-citrate solution (molar ratio 1:1, pH 3.6) and heated for 1 h at 100° C. The sample was then lyophilized and suspended in methanol. After centrifugation (7000×g for 10 min at 4° C.), lipid A was extracted with a mixture of chloroform:methanol:water (3:1.5:0.25; by volume) at a concentration of 10 μg/μl.

[0150] The molecular species present in this preparation were analyzed using an AXIMA performance matrix assisted laser desorption MALDI-TOF (matrix assisted) mass spectrometer (Shimadzu Biotech) (12BC, Université Paris Saclay, Gif sur Yvette, France). A suspension of lipid A (1 μg/μl) in chloroform:methanol:water (3:1.5:0.25, v:v:v) was desalted with a few grains of Dowex 50W-X8 (H+), 1 μl was deposited onto the target and mixed with 1 μl of a gentisic acid matrix (2,5-dihydroxybenzoic acid) (DHB from Fluka) suspended at 10 μg/μl in the same solvent or in 0.1 M of aqueous citric acid, and dried. Analyte ions were desorbed from the matrix with pulses of a 337 nm nitrogen laser. The spectra were obtained in negative ion mode at 20 kV, with the linear detector. Mass calibration was carried out with the AB SCIEX peptide mass standard kit or with a purified LPS sample and characterized by the structure of Bordetella pertussis and E. coli J5.

[0151] Like lipid A from other Gram-negative bacteria, the lipid A fragments isolated from the twelve selected strains of E. cloacae contained multiple molecular variants represented by multiple peaks in their mass spectra. Among the twelve lipids A, one of the most heterogeneous was that isolated from strain H7i (profile E with inducible cephalosporinase), with more than 13 significant peaks (13 molecular species) in its lipid A spectrum (FIG. 1). The spectrum contains a series of peaks (1360-1388, 1570-1598, 1797-1825, 1928-1956, 2035-2063) with a 28 mu distance between peaks, suggesting the presence of fatty acids of different lengths of two carbon atoms in the molecular variants of this lipid A. The composition of the molecular species corresponding to the various peaks is shown in Table 2.

TABLE-US-00002 TABLE 2 Peaks (calculated m/z) Constituents 1388.7 1599.1 1717.4 1745.5 1769.3 1783.4 1797.4 1811.4 1813.4 Total fatty acids 4 5 6 C12 1 2 1 1 1 C13 1 1 C14 1 2 1 2 1 1 C16 3OH—C14 3 3 4 4 4 4 4 4 4 2OH—C14 1 Phosphate 2 2 1 1 2 2 2 2 2 L-Ara4N Peaks (calculated m/z) Constituents 1825.4 1841.4 1877.4 1905.4 1928.5 1956.6 2035.8 2063.9 2143.8 Total fatty acids 7 C12 1 1 1 C13 C14 2 1 1 2 1 2 1 2 2 C16 1 1 1 3OH—C14 4 4 4 4 4 4 4 4 4 2OH—C14 1 Phosphate 2 2 3 3 2 2 2 2 3 L-Ara4N 1 1

[0152] The base peak at m/z=1825 is due to a bisphosphorylated glucosamine disaccharide backbone substituted with two myristic fatty acids and four hydroxymyristic fatty acids (identified as 3OH—C14 by GC-MS). This means that in the homologous peak of the corresponding doublet, at m/z=1797, one of the two myristic acids (C14:0) is replaced by lauric acid (C12:0). The following doublets (1928-1956 and 2035-2063) are explained by the addition of palmitate (C16:0) and 4-amino-4-deoxy-L-arabinose (L-Ara4N), respectively, at m/z 1797 and 1825. In addition to the two dominant peaks of the spectrum (at m/z 1797.4 and 1825.4), small adjacent peaks at +16 Da (m/z 1813.4 and 1841.4, labeled • in FIG. 1) indicate the presence of another less abundant hydroxymyristate residue. GC/MS analysis (FIG. 4) indicated the presence in this LPS of traces of α-hydroxymyristic acid (2OH—C14), thus suggesting that a species of lipid A contains this fatty acid and explaining the small size of the observed peaks. This can be produced by an ortholog of the dioxygenase LpxO identified in S. enterica serovar typhimurium. This enzyme generates 2-hydroxymyristate by hydroxylation of the myristate transferred to lipid A by the acyltransferase MsB/LpxM17. Another small peak (m/z 1769.3) in this spectrum (shown by .diamond-solid. in FIG. 1) can be explained by the presence of a minor species containing two C12:0 secondaries instead of C12+C14 (m/z 1797.4) or C14.+C14 (m/z 1825.4) present in the major species.

[0153] Position of the Lipid A Aminoarabinose and Phosphate Residues.

[0154] Monophosphorylated species of lipid A can be produced by acid hydrolysis (0.1 M HCl for 10 min at 100° C.). Labile bonds such as pyrophosphates and the acetal bond of the proximal glucosamine (phosphate bonded to C1) are hydrolyzed under these conditions. After such treatment of the lipid A of E. cloacae, peaks corresponding to bisphosphorylated and hexa-acylated species containing aminoarabinose (m/z 1928.5 and 1956.5) were completely absent from the MALTI-TOF spectrum (FIG. 2), such that the L-Ara4N group was not located on P4′ in the untreated bisphosphorylated species, because the L-Ara4N phosphate and phosphate bonds.fwdarw.4′-GlcN are both resistant. The loss of phosphoryl-aminoarabinose by mild acid hydrolysis proves that the L-Ara4N substituent is on the P1 phosphate of the bisphosphorylated species.

[0155] With respect to the phosphate groups, the presence of triphosphorylated molecules in untreated lipid A (m/z 1877.4 and 1905.4) indicates that a pyrophosphate must be present in these two molecular species. However, it should be noted that the molecules containing pyrophosphate (m/z 1877.4 and 1905.4) do not contain L-Ara4N and the molecules containing L-Ara4N (m/z 1928.5 and 1956.5) do not contain pyrophosphate. This suggests that during biosynthesis of the lipid A of E. cloacae, a third phosphate group or an L-Ara4N group is added to the P1 phosphate. It should be noted that the mild hydrolysis procedure used in this case did not induce extensive fatty acid cleavage because the hepta-acylated and monophosphorylated species (m/z 1955.8 and 1983.9) were still present after this hydrolysis.

[0156] Analysis of the Lipid A Acylation Profiles.

[0157] The sequential release of fatty acids linked to esters by mild alkaline treatment, used in previous studies (Silipo, A., et al. J. Lipid Res. 43, 2188-2195, 2002) generally provides valuable information concerning the positions of the various fatty acids on the lipid A backbone.

[0158] According to these studies, the secondary fatty acids of the C2′ and C2 groups (in particular C2′) are the most resistant, those of the C3 group (primary or secondary) are the most labile, with the substitutions of the C3′ group exhibiting intermediate behaviors. In addition, the secondary acids are more resistant than the primary fatty acids to alkaline treatments. In order to analyze the fatty acid acylation profiles in the LPS of E. cloacae, lipid A H7i was treated with 28% NH.sub.4OH at 50° C. for 30 min, 1 h or 3 h.

[0159] After this step, the complete structures of the main molecular species present in the lipid A complex of E. cloacae can now be proposed (FIG. 3). Four major and structurally different molecular species are present. In two of them (general structure A), a C14:0 secondary is at position 2, while in the other two (general structure B) it is at position 3′. Additionally, when present as a minor substituent, a palmitic group (C16:0) is at position 3′ in structure A and at position 2 in structure B.

[0160] Similarities and Differences Between the Lipid A Spectra of the Selected E. cloacae Strains. Comparison of the Lipids.

[0161] The spectra of the twelve strains of E. cloacae selected during this first step indicate that some peaks are not always present and that small additional peaks are sometimes detectable (Table 3).

TABLE-US-00003 TABLE 3 Source of isolation Cavum Blood Strain designation C17 C18 C16 C12 H1 H2 H11 H9 H8 H7o H7i H10 ERIC-PCR profile E F G H A A A B C E E F Expression AmpC* i i i i o o o i i o I i m/z (calculated) Peaks present in the spectrum 1388.7 + + + + + 1599.1 + 1717.4 + + + + 1745.5 + + + + + + + + + 1769.3 + + + + + + 1783.4 + 1797.4 + + + + + + + + + + + + 1811.4 + + + 1813.4 + + + 1825.4 + + + + + + + + + + + + 1841.4 + + + + 1877.4 + + + + + + + 1905.4 + + + + + + + + + + 1928.5 + + + + + + + + + 1956.6 + + + + + + + + 2035.8 + + + + + + + + + 2063.9 + + + + + + + + + + + + 2143.8 + + +

[0162] A tetraacylated (three hydroxymyristic acids and one myristic acid) and bisphosphorylated (peak at m/z=1388.7) glucosamine disaccharide is present in five strains (C17, C12, H8, H7o and H10), but it is absent from the other seven.

[0163] The tetraacylated molecular species observed in these five strains of E. cloacae contains three 3OH—C14 and one C14:0. The formation of this tetra-acylated species is most likely due to the loss, by enzymatic cleavage, of a myristoxy-myristoyl residue from the hexa-acylated form of lipid A at m/z 1825.4. Such cleavage requires the general structure B displayed in FIG. 3, which carries a C14:0 secondary on 3′. No correlation was found between this enzymatic cleavage detected in only five strains (C17, C12, H8, H7o and H10) and the ERIC-PCR profiles of these strains (profiles E, H, C, E and F, respectively).

[0164] The presence of other small peaks represents a second type of structural variation between the twelve strains. The small peaks at m/z 1813.4 and 1841.4 mentioned above in the spectrum of the H7i strain (presence of an α-hydroxymyristic acid) were actually present in four strains: H1, H7o, H7i and H10 (Table 3). They are due to a 2-hydroxymyristic acid (2OH—C14) that replaces the C14:0 secondary at positions 2 (FIG. 3A) or 3′ (FIG. 3B). The same C14:0 can also be replaced by C13:0 (peak at 1811.4 in strains C12, H2 and H8) or by C12:0 (peak at m/z 1769.3 in strains C17, C18, C12, H8, H7o and H7i) (Table 3 and FIG. 3).

[0165] Fatty Acid Composition of the LPSs Analyzed by Gas Chromatography/Mass Spectrometry.

[0166] The LPS samples (200 μg) were incubated at 85° C. for 15 to 18 h (but this time can be significantly reduced) in 600 μl of an anhydrous methanol/acetyl chloride mixture (10/1.5 in volumes) containing 4 μg of arachidic (eicosanoic) acid (C20) used as an internal standard. After this transmethylation reaction, the methanol was evaporated in a vacuum at room temperature, the resulting fatty acid methyl esters were extracted in ethyl acetate (600 μl) and the solvent was evaporated at room temperature under a stream of nitrogen. The material was dissolved in 50 μl of ethyl acetate and the solution (1-5 μl) was analyzed by gas chromatography coupled with mass spectrometry (GC-MS) in a Shimadzu appliance (GCMS-QP2010SE). A Phenomenex capillary column (Zebron ZB-5MS, 30 m<0.25 mm<0.25 μm) was used with a temperature gradient of 50° C. to 120° C. (20° C./min) followed by a gradient from 120° C. to 250° C. (3° C./min) and finally a constant temperature (250° C. for 2 min). The fatty acids were identified by their mass spectra (NIST base) and their retention times compared to the fatty acid standards (Sigma-Aldrich).

[0167] Analysis of the fatty acid composition of the LPSs by gas chromatography/mass spectrometry (GC/MS) confirmed the results obtained by MALDI, in particular the presence of small amounts of tridecanoic and 2-hydroxymyristic groups in some LPSs (FIG. 4).

[0168] Traces of a 2-hydroxylauric group (2OH—C12), undetectable by MALDI, were also detected by GC/MS in the LPSs of strains C12, H1, H9, H7o, H7i and H10.

[0169] Further variations among the twelve isolates are produced by the absence of significant MALDI peaks characteristic of the most complete molecular species. This is the case for the absence of pyrophosphate (trisphosphoryl species at m/z 1877.4 and 1905.4) in strains C17 and H11, as well as the absence of L-Ara4N (peaks at m/z 1928.5 and 1956.6) in strains C17, C16 and C12 (Table 2).

[0170] No correlation was observed between the lipid A structures of the various strains, characterized by their mass spectra, and their other available characteristics, such as their ERIC-PCR profile (A to H), the source from which they were isolated (cavum, rectum or blood), or the expression of cephalosporinase (inducible or overproduced). For example, strains H7o and H7i (isolated from the same infant) have the same ERIC-PCR profile and very similar lipid A mass spectra, but differ in terms of the cephalosporinase expression. Conversely, strains H11 and H7o both have an overproduction of cephalosporinase, but exhibit different lipid A spectra and ERIC-PCR profiles (profiles A and E, respectively).

Example 2: Correlation Between the Presence of 2-Hydroxymyristate and the Pathogenicity of the Strains of the E. cloacae Complex

[0171] Although all the selected strains carry the same major molecular species of lipid A, they differ in minor variations, such as the presence of an L-Ara4N or a pyrophosphate at position 1 and the presence of a 2-hydroxymyristate (2OH—C14) or a C13:0 replacing a myristate (FIG. 3). It is therefore possible to classify the strains as a function of the presence of molecular species in their lipid A that may or may not carry some of these four constituents. Using this method, the twelve strains can be grouped into six groups as shown in Table 4.

TABLE-US-00004 TABLE 4 L-Ara4N+/PP+ L-Ara4N+/P− L-Ara4N−/PP+ 2OH-C14+/C13− H1 (†); H7h (†) H7i (†); H10 (†) 2OH-C14-/C13+ H2; H8 (†) C12 2OH-C14−/C13− H9; C18 H11 C17; C16 †: deceased infant; L-Ara4N: 4-amino-4-deoxy-L-arabinose; PP: pyrophosphate; 2OH-C14: 2-hydroxymyristic acid; C13: tridecanoic acid. ″+″ indicates the presence and ″−″ the absence of the corresponding substituent.

[0172] Notably, the lethal strains are not randomly distributed in the 6 identified groups but belong to only two groups. This result may suggest that the presence of 2-hydroxymyristate (2OH—C14) (100% (3/3) death) and, to a lesser extent, of tridecanoate (C13) (1/3 death; 33%) clearly improves the pathological power of the strain compared to the strains without these fatty acids (0% death).

[0173] A larger number of bacteraemic and non-bacteraemic babies was then studied to verify the correlation between the lethality induced by these strains in these two population groups and a structural element of their LPS system. These two population groups of infants were selected to be homogeneous in terms of their other characteristics and, in particular, do not differ in terms of other risk factors that contribute to neonatal mortality but are not related to their bacterial carriage. To this end, the CRIB II index (for “Clinical Risk Index for Babies”) was used. The CRIB II index is a hospital grading system that is widely used in neonatal intensive care and that reflects the severity of the condition of hospitalized babies. The score takes into account several characteristics of newborns, including the birth weight, the sex, the gestational age, the temperature, the Apgar score at 5 min, the base excess, the maternal age and parity.

[0174] In order to obtain a homogeneous population of infants, 18 premature infants infected with the E. cloacae complex were first selected. Then, for each of these babies, a colonized infant with a similar CRIB II score was selected. Strains from 18 infected infants were isolated from the blood culture, while in 18 colonized infants, 9 strains were isolated from the cavum and 9 from the rectum (Table 5). Of the 36 infants, 13 died within one month of birth, including 12/18 in the group of infected infants and 1/18 in the group of colonized infants. Death occurred on average 10±8 days after birth.

[0175] The presence of 2-hydroxymyristate (2OH—C14) in the LPS, as determined by GC-MS, was associated with the detection of the IpxO gene in 8 strains (Table 5). In LPS H9, 2-hydroxymyristate (2OH—C14) was undetectable in the MALDI spectrum (absence of peaks at m/z 1813.4 and 1841.4) and almost absent by GC-MS analysis (0.5%) compared with the average amount of this constituent in other strains (4.3±1.4%). It was therefore considered that LPS H9 is devoid of 2OH—C14. Data relating to the number of deaths as a function of the presence or absence of 2OH—C14 in the corresponding LPSs was extracted from Table 5.

TABLE-US-00005 TABLE 5 Strain Expression IpxO Death of Status of Source of of AmpC 2OH—C14 gene the baby the baby isolation Designation * (%) ** *** **** colonized Cavum C1 i 6.3 + + C2 i 0 − − C3 i 0 − − C4 i 0 − − C6 i 0 − − C7 i 0 − − C9 i 0 − − C21 i 0 − − C25 i 0 − − Rectum C5 o 0 − − C8 o 0 − − C10 o 0 − − C11 o 0 − − C23 o 4.7 + − C24 o 4.9 + − C26 o 0 − − C27 i 0 − − C28 i 0 − − infected blood H1 o 6.3 − + H2 o 0 − − H3 o 2.6 − + H4 o 5.4 + + H5 i 4.2 + + H6 o 0 − + H7 o 4.9 + + H8 i 0 − + H9 i 0.5 − − H10 i 4.5 + + H11 o 0 − − H21 i 0 − − H23 i 0 − + H24 o 0 − + H25 i 0 − − H26 o 2.2 + − H27 i 0 − + H28 i 0 − + * inducible (i) or overproduced (o) cephalosporinase. ** expressed by the ratio: 2OH—C14 × 100/3OH—C14. *** + detected; − not detected. **** + baby died within the first month after birth; − baby survived.

[0176] Patients with E. cloacae sepsis carrying this 2OH—C14 marker in the LPS were compared with those lacking the marker. Patients with LPS carrying 2OH—C14 have a higher VIS score (vasoactive and inotropic drugs needed to maintain normal systemic blood pressure) (p=0.03) and are more likely to develop oliguria (p=0.02) than those without this LPS marker. A Fisher's exact test carried out on the data shows a significant dependence (p=0.0178) between the presence of 2OH—C14 in the LPSs of the E. cloacae complex and the mortality induced by these strains.

[0177] The deaths of 9 newborns were directly attributable to E. cloacae septic shock. Other causes of death were spontaneous intestinal perforation (n=2) and Haemophilus influenzae pneumonia (n=1), Bacteroides fragilis infection (n=1), necrotizing enterocolitis (n=1), and severe bronchopulmonary dysplasia (n=1). Consequently, the data corresponding to deaths not attributable to E. cloacae septic shock must be removed from the statistical analysis. A survival analysis according to Kaplan-Meier shows a highly significant imputability on mortality of the presence of LPS carrying 2OH—C14 (p=0.007 using the logrank test).

[0178] The results are shown in FIG. 5. On the diagram, the solid line curve corresponds to the strains for which 2-hydroxymyristic acid is present in the LPS. The dashed curve corresponds to the strains for which 2-hydroxymyristic acid is absent in the LPS.

[0179] The risk ratio of death in patients infected with Enterobacter cloacae whose LPS has 2OH—C14 is 5.55.

Example 3: E. cloacae Complex Polymyxin B Survival Assay

[0180] Strains of the Enterobacter complex were selected.

[0181] The Enterobacter strains preserved in a tube frozen with 30% glycerol are transplanted in a planktonic culture in 20 ml of an LB culture medium. After incubation at 37° C. overnight, 1 ml of the culture diluted to 10.sup.7 is brought into contact with increasing concentrations of polymyxin B (1 μg/ml, 3 μg/ml and 10 μg/ml). A tube without antibiotic is added as a control.

[0182] All the tubes are incubated at 37° C. with shaking for an additional 4 hours. The cultures are then diluted and spread on LB agar Petri dishes.

[0183] The dishes are incubated at 37° C. overnight and the colonies are counted.

[0184] Percentage survival is computed as follows:

(CFU polymyxin B/CFU control)×100

[0185] 5 strains of E. cloacae complex expressing 2-hydroxymyristate (2HM) and 5 strains of E. cloacae complex not expressing 2-hydroxymyristate (2HM) were exposed to different concentrations of polymyxin B for 4 hours.

[0186] The results presented in FIG. 6 show that the presence of 2-OH myristate on Lipid A generates resistance to treatment with polymixin B.

Example 4: Study of the Presence of lpxO

[0187] The insertion of 2-hydroxymyristate (2HM) into the Lipid A structure is under the control of an LpxO hydroxylase described in Salmonella. The lpxO gene exists within the bacterial chromosome sequences of several ECCs on GenBank.

[0188] Whole genome sequencing of the 20 strains of Enterobacter cloacae complex (ECC) was carried out using Illumina technology. An alignment of sequences via Clustal Omega between the lpxO sequences of our strains (n=5) and sequences available on GenBank from among the genomes of ECC and other Gram-negative bacteria was carried out. The search in the NGS sequences for a counterpart of lpxO found its presence in 5 of the 7 strains with 2HM. Within the genus Enterobacter, the lpxO sequences of the various species are more than 80% identical except for all the sequences available in E. hormaechei, which had only 60% identity with the other Enterobacter. While the lpxO sequences from the same Enterobacter species vary very little (for example, from 96% to 100% in E. bugandensis and from 99 to 100% in E. cloacae), all the lpxO sequences of E. hormaechei are 100% identical and appear to originate from a plasmid.