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• W3187992502 abstract "•Fifteen different molecular species of lipid A is found in E. cloacae complex•2-Hydroxymyristate moiety on Lipid A is a virulence marker of the E. cloacae complex•Presence of 2-hydroxymyristate is associated with mortality in neonatal sepsis Enterobacter cloacae complex species are involved in infections among critically ill patients. After a recent E.cloacae outbreak of fulminant neonatal septic shock, we conducted a study to determine whether septic shock severity and its lethal consequence are related to structural features of the endotoxin (lipopolysaccharide [LPS]) of the strains isolated from hospitalized infants and more specifically its lipid A region. It appeared that the LPSs are very heterogeneous, carrying fifteen different molecular species of lipid A. The virulence was correlated with a structural feature identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and gas chromatography coupled with mass spectrometry: the presence of 2-hydroxymyristic acid as a secondary substituent in lipid A. This is the first published evidence linking LPS structural moiety to neonatal sepsis outcome and opens the possibility of using this fatty acid marker as a detection tool for high-risk patients, which could help reduce their mortality. Enterobacter cloacae complex species are involved in infections among critically ill patients. After a recent E.cloacae outbreak of fulminant neonatal septic shock, we conducted a study to determine whether septic shock severity and its lethal consequence are related to structural features of the endotoxin (lipopolysaccharide [LPS]) of the strains isolated from hospitalized infants and more specifically its lipid A region. It appeared that the LPSs are very heterogeneous, carrying fifteen different molecular species of lipid A. The virulence was correlated with a structural feature identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and gas chromatography coupled with mass spectrometry: the presence of 2-hydroxymyristic acid as a secondary substituent in lipid A. This is the first published evidence linking LPS structural moiety to neonatal sepsis outcome and opens the possibility of using this fatty acid marker as a detection tool for high-risk patients, which could help reduce their mortality. Species of a Enterobacter cloacae complex, gram-negative bacteria of the Enterobacteriaceae family, are commonly isolated from soil, plants, and from the digestive tract of mammals and insects [Jang and Nishijima, 1990Jang E.B. Nishijima K.A. Identification and attractancy of bacteria associated with Dacus dorsalis (Diptera, Tephritidae).Environ. Entomol. 1990; 19: 1726-1731Crossref Scopus (54) Google Scholar; Marchini et al., 2002Marchini D. Rosetto M. Dallai R. Marri L. Bacteria associated with the oesophageal bulb of the medfly Ceratitis capitata (Diptera : tephritidae).Curr. Microbiol. 2002; 44: 120-124Crossref PubMed Scopus (58) Google Scholar]. As a facultative anaerobe, it has the ability to survive in various environments, including dry soil, water pipes, and metal or plastic medical equipment [Herson et al., 1987Herson D.S. McGonigle B. Payer M.A. Baker K.H. Attachment as a factor in the protection of Enterobacter cloacae from chlorination.Appl. Environ. Microbiol. 1987; 53: 1178-1180Crossref PubMed Scopus (78) Google Scholar]. The E. cloacae complex is a common catheter contaminant [Watson et al., 2005Watson J.T. Jones R.C. Siston A.M. Fernandez J.R. Martin K. Beck E. Sokalski S. Jensen B.J. Arduino M.J. Srinivasan A. Gerber S.I. Outbreak of catheter-associated Klebsiella oxytoca and Enterobacter cloacae bloodstream infections in an oncology chemotherapy center.Arch. Intern. Med. 2005; 165: 2639-2643Crossref PubMed Scopus (40) Google Scholar; Harbarth et al., 1999Harbarth S. Sudre P. Dharan S. Pittet M. Pittet D. Outbreak of Enterobacter cloacae related to understaffing, overcrowding, and poor hygiene practices.Infect. Control Hosp. Practices. 1999; 20: 598-603Crossref PubMed Scopus (256) Google Scholar] and can be an opportunistic pathogen of immunocompromised adults and neonates [Mayhall et al., 1979Mayhall C.G. Lamb V.A. Gayle Jr., W.E. Haynes Jr., B.W. Enterobacter cloacae septicemia in a burn center: epidemiology and control of an outbreak.J. Infect. Dis. 1979; 139: 166-171Crossref PubMed Google Scholar; Davin-Regli et al., 2019Davin-Regli A. Lavigne J.P. Pagès J.M. Enterobacter spp./Update of Taxonomy, Clinical aspects, and emerging antimicrobial resistance.Clin. Microbiol. Rev. 2019; 32 (e00022–19)Crossref PubMed Scopus (63) Google Scholar]. The E. cloacae complex includes various species where Enterobacter cloacae, Enterobacter bugandensis, and Enterobacter hormaechei represent the most frequently isolated species in clinical infections, especially in the neonatal intensive care unit (NICU) [Davin-Regli et al., 2019Davin-Regli A. Lavigne J.P. Pagès J.M. Enterobacter spp./Update of Taxonomy, Clinical aspects, and emerging antimicrobial resistance.Clin. Microbiol. Rev. 2019; 32 (e00022–19)Crossref PubMed Scopus (63) Google Scholar; Pati et al., 2018Pati N.B. Doijad S.P. Schultze T. Mannala G.K. Yao Y. Jaiswal S. Ryan D. Suar M. Gwozdzinski K. Bunk B. et al.Enterobacter bugandensis: a novel enterobacterial species associated with severe clinical infection.Sci. Rep. 2018; 8: 5392Crossref PubMed Scopus (22) Google Scholar]. In humans, the E. cloacae complex is a member of the normal gut microbiota [Keller et al., 1998Keller R. Pedroso M.Z. Ritchmann R. Silva R.M. Occurrence of virulence-associated properties in Enterobacter cloacae.Infect. Immun. 1998; 66: 645-649Crossref PubMed Google Scholar]. In recent years, the E. cloacae complex has emerged as one of the most commonly found nosocomial pathogen in the NICU, but little is known and has been published about its virulence-associated factors [Dalben et al., 2008Dalben M. Varkulja G. Basso M. Krebs V.L. Gibelli M.A. van der Heijden I. Rossi F. Duboc G. Levin A.S. Costa S.F. Investigation of an outbreak of Enterobacter cloacae in a neonatal unit and review of the literature.J. Hosp. Infect. 2008; 70: 7-14Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar]. The aim of this study was to search for an eventual correlation between lipopolysaccharides (LPS) structure and the pathogenicity of strains of the E. cloacae complex isolated in premature infants with septic shock. Endotoxins (LPS) are recognized virulence factor by interfering with host recognition, immune response, and action of antimicrobial agents. Modifications of the lipid A region of the LPS molecules are known to modify the penetration ability of some antimicrobial agents [Nikaido, 2003Nikaido H. Molecular basis of bacterial outer membrane permeability revisited.Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2544) Google Scholar]. Similarly, an increased virulence of Gram-negative bacteria can be due to increased resistance to different host cationic antimicrobial peptides (CAMPs) such as defensins, human neutrophil peptide (HNP-1), human cationic protein 18 (hCAP18 or LL-37), and the human platelet-derived kinocidin [Peschel and Sahl, 2006Peschel A. Sahl H.G. The co-evolution of host cationic antimicrobial peptides and microbial resistance.Nat. Rev. Microbiol. 2006; 4: 529-536Crossref PubMed Scopus (736) Google Scholar]. The main mechanisms of resistance to CAMPs consist of LPS modification through the addition of 4-amino-4-deoxy-L-arabinose or phosphoethanolamine, which decreases the negative charge of the lipid A. The operons encoding enzymes involved in these modifications are arnBCADTEF and pmrCAB, respectively [Olaitan et al., 2014Olaitan A.O. Morand S. Rolain J.M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria.Front. Microbiol. 2014; 5: 643Crossref PubMed Google Scholar]. Activation of the LPS-modifying genes is often mediated through PmrA/PmrB and PhoP/PhoQ, two-component regulatory systems that are interconnected depending on the species. For instance, in Escherichia coli, Salmonella enterica, or Klebsiella pneumoniae, the phosphorylated form of PhoP can stimulate the expression of PmrD that in turn activates PmrA promoting the transcription of the arnBCADTEF and pmrCAB operons. Regarding the E. cloacae complex and its resistance to CAMPs, it has been shown recently that the arn operon is involved, but in contrast to E. coli, Salmonella, or Klebsiella, only PhoP/PhoQ (and not PmrA/PmrB) seems to play a role [Gibbons et al., 2000Gibbons H.S. Lin S. Cotter R.J. Raetz C.R. Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A: function of LpxO, a new Fe2+/α-ketoglutarate-dependent dioxygenase homologue.J. Biol. Chem. 2000; 275: 32940-32949Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar]. An E. cloacae complex outbreak occurred in the NICU of Antoine Béclère Hospital (Clamart, France). The “fulminating” course of nosocomial infection due to the E. cloacae complex in critically ill patients urges to investigate the role of endotoxins in the pathophysiology of those infections. Suggested by the fulminating and devastating course of septic shock, we hypothesized that LPSs structures of the patient's isolated E. cloacae complex strains, particularly their lipid A moiety, might display some specific structural signatures associated with this severe pathophysiologic response. From January 2016 to June 2017, 18 patients admitted in the NICU of the Antoine Béclère Hospital (AP-HP Paris-Saclay University, Clamart, France) had E. cloacae complex septic shock. Patient characteristics are displayed in Table S1. All patients were extremely premature infants (median 27.0 weeks of gestational age). Of the 18 infants, 12 infants displayed a fulminant course with death occurring within a median of 61 h (IQR 22–1062) after septic shock diagnosis. Death occurred on a median of 7 days (IQR 4–10) after birth. Nineteen E. cloacae complex strains were isolated from blood cultures. For one patient, we isolated two E. cloacae complex strains with different antibiotic-resistant pattern (H7i and H7o). Among the 19 E. cloacae complex isolates, we identified E. hormaechei (n = 11), E. bugandensis (n = 7), and E. cloacae (n = 1) (Table S2). We identified 9 enterobacterial repetitive intergenic concensus (ERIC)–polymerase chain reaction (PCR) profiles (Figure S1). In E. hormaechi species, 5 profiles were identified with a majority of the profile A (7 of 11, 63.7%). In E. bugandensis species, 4 profiles were identified with a majority of the profile E (4 of 7, 57.1%). Among all E. cloacae complex strains, we detected an overproduction of AmpC cephalosporinase contributes to the resistance to third-generation cephalosporins in 8 of 19 strains (42.1%). Phenotypic antibiotic resistance patterns were not evenly distributed among E. cloacae complex species. The species E. hormaechei showed a higher resistance to third-generation cephalosporins (6 of 11, 54.5%) compared to other species (2 of 8, 25.0%) (Table S2). LPSs were extracted from the cultured strains and the structures of their lipid A regions were analyzed by matrix-assisted laser desorption ionization–time of flioght (MALDI-TOF). Like lipid A of other Gram-negative bacteria, the lipid A moieties isolated from the twelve selected strains of the E. cloacae complex contained multiple molecular variants represented by multiple peaks in their mass spectra. Among the twelve lipid A, one of the more heterogeneous was that isolated from strain H7i (E. bugandensis E profile with low level of a chromosomal AmpC beta-lactamase), with more than 13 significant peaks (13 molecular species) in its lipid A spectrum (Figure 1). The spectrum contained a series of peaks (1360–1388, 1570–1598, 1797–1825, 1928–1956, 2035–2063) with an interpeak distance of 28 mu, suggesting that fatty acids of length differing by two carbons were present in molecular variants of this lipid A. The composition of the molecular species corresponding to the different peaks is indicated in Table 1. The base peak was at m/z = 1825 and was likely due to a bisphosphorylated glucosamine disaccharide backbone substituted with two myristic and four hydroxymyristic fatty acids (identified as 3OH-C14 by gas chromatography coupled with mass spectrometry [GC-MS]). This meant that in the homologous peak of the corresponding doublet, at m/z = 1797, one of the two myristic (C14:0) acids was replaced with a lauric (C12:0) fatty acid. The following doublets (1928–1956 and 2035–2063) could be easily explained by the addition of palmitate (C16:0) and 4-amino-4-deoxy-L-arabinose (L-Ara4N), respectively, to the m/z 1797 and 1825 molecular species.Table 1Composition of molecular species detectable by MALDI-TOF in Lipid A of E. cloacae complexConstituentsPeaks (calculated m/z)1388.71599.11717.41745.51769.31783.41797.41811.41813.41825.41841.41877.41905.41928.51956.62035.82063.92143.8Total fatty acids4567C1212111111C1311C14121211211212122C161113OH-C143344444444444444442OH-C1411Phosphate221122222223322223L-Ara4N11The figures shown represent the number of each residue present in the molecular species characterized by a particular mass peak. Open table in a new tab The figures shown represent the number of each residue present in the molecular species characterized by a particular mass peak. In addition to the two dominant peaks of the spectrum (at m/z 1797.4 and 1825.4), small flanking peaks at +16 Da (m/z 1813.4 and 1841.4, marked • in Figure 1) indicating the presence of an alternative hydroxymyristate residue which was used at low frequency in place of C14:0 by a variant acyltransferase. The analysis by GC-MS (Figure 2) indicated the presence in this LPS of trace amounts of α-hydroxymyristic acid (2OH-C14), thus suggesting that a species of lipid A contained this fatty acid and accounted for the small flanking peaks observed. This can be produced by an ortholog of the dioxygenase LpxO identified in Salmonella enterica serovar Typhimurium [Gibbons et al., 2000Gibbons H.S. Lin S. Cotter R.J. Raetz C.R. Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A: function of LpxO, a new Fe2+/α-ketoglutarate-dependent dioxygenase homologue.J. Biol. Chem. 2000; 275: 32940-32949Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar]. In silico analyses of several E. cloacae complex genomes revealed that this pathogen carries one homolog of known lpxO in others Gram-negative bacteria. Blast analysis of E. cloacae strain ATCC 13047 showed that LpxO (GenBank: NC_014121) is 84%, 63%, 57%, 56%, and 57% identical to S. enterica serovar Typhimurium, K. pneumoniae, Burkholderia pseudomallei, Acinetobacter baumannii, and Pseudomonas aeruginosa LpxO, respectively (Figure 3). Among E. cloacae complex species, E. cloacae LpxO shared 90% and only 67% sequence identity with E. bugandensis (GenBank: NZ_POUP01000001) and E. hormaechei LpxO (GenBank: NZ_NJCO01000003), respectively. LpxO is known to generate 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 (marked ⧫ in Figure 1) can be explained by the presence of a minor species containing two secondary C12:0 instead of the C12 + C14 (m/z 1797.4) or C14 + C14 (m/z 1825.4) present in the major species. Position of the aminoarabinose and phosphate residues of the lipids A. Monophosphorylated species of lipid A can be produced by acid hydrolysis (0.1 M HCl for 10 min at 100°C). Labile linkages such as pyrophosphates and the acetal linkage of the proximal glucosamine (phosphate linked to C1) are hydrolyzed under these conditions. After such a treatment of E. cloacae complex lipid A, the peaks corresponding to the bisphosphorylated and hexa-acylated species containing aminoarabinose (m/z 1928.5 and 1956.5) were completely absent from the MALTI-TOF spectrum (Figure 4). Therefore, the L-Ara4N group was not located on P4′ in the untreated bisphosphorylated species because the L-Ara4N→phosphate and the phosphate→4′-GlcN linkages are both resistant to this moderate acid hydrolysis. The loss of phosphoryl-aminoarabinose by mild acid hydrolysis proved that the L-Ara4N substituent was on phosphate at position 1 of the bisphosphorylated species.Figure 3E. cloacae complex LpxO homologs in Gram negative bacteriaShow full captionIn silico analysis revealed that E. cloacae complex carries one homolog of known lpxO in others Gram-negative bacteria. LpxO homology between E. cloacae (GenBank: NC_014121), S. enterica serovar typhimurium (GenBank: NZ_CP043907), K. pneumoniae (GenBank: NZ_FO834906), B. pseudomallei (GenBank: NC_017832), A. baumannii (GenBank: NZ_CP059041), P. aeruginosa (GenBank: NZ_CP017149), E. bugandensis (GenBank: NZ_POUP01000001), and E. hormaechei (GenBank: NZ_NJCO01000003).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Negative-ion MALDI/TOF mass spectrometry of E. cloacae complex H7i (E. bugandensis ERIC-PCR Profile low level of chromosomal AmpC beta-lactamase) lipid A, untreated or hydrolyzed with HCl for 10 min at 100°CShow full captionP, L-Ara4N and C16 represent m/z shifts corresponding to phosphate, 4-amino-4-deoxy-L-arabinose and palmitate substituents, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In silico analysis revealed that E. cloacae complex carries one homolog of known lpxO in others Gram-negative bacteria. LpxO homology between E. cloacae (GenBank: NC_014121), S. enterica serovar typhimurium (GenBank: NZ_CP043907), K. pneumoniae (GenBank: NZ_FO834906), B. pseudomallei (GenBank: NC_017832), A. baumannii (GenBank: NZ_CP059041), P. aeruginosa (GenBank: NZ_CP017149), E. bugandensis (GenBank: NZ_POUP01000001), and E. hormaechei (GenBank: NZ_NJCO01000003). P, L-Ara4N and C16 represent m/z shifts corresponding to phosphate, 4-amino-4-deoxy-L-arabinose and palmitate substituents, respectively. Regarding phosphate groups, the presence of triphosphorylated molecules in the untreated lipid A (m/z 1877.4 and 1905.4) indicates that a pyrophosphate must be present in these two molecular species. However, we can note that molecules containing pyroposphate (m/z 1877.4 and 1905.4) do not contain L-Ara4N, and molecules containing L-Ara4N (m/z 1928.5 and 1956.5) do not contain a pyrophosphate. This suggests that during the biosynthesis of the E. cloacae complex lipid A, either a third phosphate group or an L-Ara4N group is added on phosphate at position 1. It is noteworthy that the moderate hydrolytic procedure used here did not induce important cleavage of fatty acids since the hepta-acylated and monophosphorylated species (m/z 1955.8 and 1983.9) were still present after this hydrolysis. The sequential liberation of ester-linked fatty acids by mild alkaline treatment, as used in previous studies [Olaitan et al., 2014Olaitan A.O. Morand S. Rolain J.M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria.Front. Microbiol. 2014; 5: 643Crossref PubMed Google Scholar; Silipo et al., 2002Silipo A. Lanzetta R. Amoresano A. Parrilli M. Molinaro A. Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution.J. Lipid Res. 2002; 43: 2188-2195Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar], usually provides valuable information on the positions of the different fatty acids on the lipid A backbone. According to these studies, the more resistant are the secondary fatty acids on C2′ and C2 (particularly C2′), and the more labile are those on C3 (either primary or secondary), substitutions on C3′ showing intermediate behaviors. In addition, secondary fatty acids are more resistant to alkaline treatments than primary fatty acids. In order to analyze the acylation patterns of the fatty acids in E. cloacae complex LPS, we treated the H7i lipid A with 28% NH4OH at 50°C for 30 min or 3 h (Figure 5 and Figure 6). When the treatment was performed for only 30 min, new hexa-, penta-, and tetra-acylated species were produced. Important information on this lipid A structure and composition can already be gained from the group of four peaks of m/z between 1340 and 1390. The peak at m/z 1344.7 (two phosphates, two hydroxymyristates, one C12:0, and one C14:0) indicate that only C12:0 and C14:0 are the secondary substituents at 2 and 2′ of the two remaining, amide-linked, hydroxymyristate. The lauric residue C12:0 must be on C2′ because this position is the most resistant to alkaline hydrolysis and C12:0 remains present in several other de-O-acylated fragments (peaks at m/z 1797.4, 1571 and 1360.7), even after 3 h of hydrolysis (peak at m/z 1134.3). One myristic residue must also be present on the same 2′ position because it is also found after a 3-h hydrolysis (triacylated species at m/z 1162.4 with only one C14:0). Because the 2′ position is substituted by either C12:0 or C14:0, it follows that in the species at m/z 1344.7 mentioned above, the C14:0 residue is at position 2. The peak at 1372.7 represents the homologous species with secondary C14:0 on 2 and 2'. Therefore, two major molecular species of this lipid A contain a C14:0 on position 2. On the other hand, the peak at m/z 1360.7 (two phosphates, three hydroxymyristates and one C12:0) results from the easy loss of only one hydroxymyristate at position 3 (peak at m/z 1571.0) followed by the cleavage of a C14:0. Because secondary substituents at position 2 are firmly linked, the cleaved myristate was not initially at position 2 but on the only remaining position, 3'. Therefore, the peak at m/z 1360.7 containing C12:0, and its homolog at m/z 1388.7 containing C14:0, derive from two other major molecular species, different from those mentioned in the preceding paragraph, which contain a C14:0 on position 3′ and an unsubstituted primary hydroxymyristate on position 2. This means that the group of four peaks of m/z between 1340 and 1390 represent four major and structurally different molecular species present in this lipid A. In two of those (general structure A), a secondary C14:0 is at position 2, whereas in the two others (general structure B), it is at position 3' (Figure 7).Figure 6Pattern of cleavage of the molecular species present in the E. cloacae complex H7i (E. bugandensis ERIC-PCR Profile, with low level of chromosomal AmpC beta-lactamase) during alkaline treatmentShow full captionNumbers are the m/z values observed in MALDI-TOF obtained after treatment with 28% NH4OH at 50°C for 30 min or 3 hr (see Figure 5). The number of fatty acids (FA) in each species is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Structure of the major molecular species in the E. cloacae complex H7i (E. bugandensis ERIC-PCR Profile; with low level of chromosomal AmpC beta-lactamase)Show full captionStructures (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Numbers are the m/z values observed in MALDI-TOF obtained after treatment with 28% NH4OH at 50°C for 30 min or 3 hr (see Figure 5). The number of fatty acids (FA) in each species is indicated. 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. Regarding the addition of an acyl-oxo-acyl C14 at the 2 position, the observed peak at m/z 1344.7 (Figure 5) found after mild alkaline treatment (30 min NH4OH) of E. cloacae complex lipid A can only result from structure A (shown in Figure 7) after the loss of the labile substituents on C3 and C3' (see scheme in Figure 6). As mentioned above, m/z 1344.7 correspond to a molecule with a C14:0 residue linked to the C14OH at position 2 (it is not located at position 2' and would have been lost during this treatment if located elsewhere). Therefore, between the two main structures of E. cloacae lipid A, one is palmitoylated (Figure 7B) and the other is myristoylated (Figure 7A) on the C14OH at C2. In the latter case, the addition of a C14 acyl-oxy-acyl at the C2 position is rare but not novel. A similar feature has been described in Vibrio cholerae O 1 lipid A, due to the activity of MsbB [Matson et al., 2010Matson J.S. Yoo H.J. Hakansson K. Dirita V.J. Polymyxin B resistance in El Tor Vibrio cholerae requires lipid acylation catalyzed by MsbB.J. Bacteriol. 2010; 92: 2044-2052Crossref Scopus (39) Google Scholar; Tanamoto et al., 2001Tanamoto K.I. Iida T. Haishima Y. Azumi S. Endotoxic properties of lipid A from Comamonas testosteroni.Microbiology. 2001; 147: 1087-1094Crossref PubMed Scopus (4) Google Scholar]. Regarding the palmitic acid present in the hepta-acylated species of lipid A (peaks at m/z 2035.8 and 2063.9), after treatment with NH4OH for 3 h, we observe small peaks at 1372.7 and 1400.8 representing bisphosphorylated and diacylated molecules containing one C12/C14 and one palmitate (C16:0). The fact that C16:0 remains after this harsh treatment indicate that this palmitic group is on position 2. This can only occur in the general structure B because position 2 is already occupied by C14:0 in structure A. Concerning the latter, two positions are available to accommodate C16: 3 and 3'. After 30 min of NH4OH treatment, small peaks at m/z 1809.4 and 1837.5 are visible (not shown) and are attributable to hexa-acylated species containing a secondary C16:0 at position 3' (because the hydroxymyristic acid at position 3 is the absent). In conclusion, when present as a minor substituent in lipid A, C16:0 is on 3′ in structure A and on 2 in structure B. After the cleavage of ester-linked fatty acids with NH4OH for 30 min (Figure 6), a series of five small peaks attributable to bisphosphorylated molecules with one L-Ara4N (m/z 1730.2, 1702.2, 1519.8, 1491.8, and 1293.5) and to trisphosphorylated molecules without L-Ara4N (m/z 1679.1, 1651.0, 1468.6, 1242.3, and 1214.3) were visible in the spectrum (marked with ∗ and #, respectively, in Figure 4). In contrast, species with three phosphates and one L-Ara4N were not detected. This is in line with the absence of such molecules in the untreated lipid A (Figure 1) and strongly suggests that the phosphate at position 1 can be substituted either by an L-Ara4N group, or by a phosphate group, but not by a phosphoryl aminoarabinose. After this step of our analysis, the complete structures of the main molecular species present in E. cloacae complex lipid A can now be proposed (Figure 7). Four major and structurally different molecular species are present: In two of these (general structure A), a secondary C14:0 is at position 2, whereas in the two others (general structure B), it is at position 3'. In addition, when present as a minor substituent, a palmitic group (C16:0) is on position 3′ in structure A and on position 2 in structure B. Comparison of the lipid A spectrum of the twelve E. cloacae complex selected strains in this first step of our study indicated that some peaks were not always present, and additional small peaks were sometimes detectable (Table 2). A tetra-acylated (three hydroxymyristic and one myristic fatty acids) and bisphosphorylated glucosamine disaccharide (peak at m/z = 1388.7) is present in five strains (C17, C12, H8, H7o, and H10) but absent from the seven others. It should be noted that in E. coli, a tetra-acylated and bisphosphorylated GlcN-disaccharide (precursor IVA) plays an important role in the biosynthesis of the core region (attachment of two Kdo residues to precursor IVA). However, the tetra-acylated precursor IVA of E. coli contains four 3OH-C14, whereas the tetra-acylated molecular species observed in these five strains of E. cloacae complex 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 a cleavage requires the general structure B displayed in Figure 7, which carries a secondary C14:0 on 3'. No correlation was found between this enzymatic cleavage detected in only five strains (C12,C17, H7o, H8, and H10) and the ERIC-PCR profiles of these strains (profiles H, E, E,C, and F, respectively) and the identification E. cloacae complex species.Table 2Peaks present in MALDI-TOF spectra of lipid A isolated from various E. cloacae complex strainsSource of isolationCavumBloodStrain designationC17C18C16C12H1H2H11H9H8H7oH7iH10Identification speciesaN: No; Y: Yes. NP, not performed; E.h. Enterobacter hormaechei; E.b, Enterobacter bugandensis.NPNPNPNPE.hE.hE. hE.bE.hE.bE.bE.bERIC-PCR profileEFGHAAABCEEFOverproduction of AmpCaN: No; Y: Yes. NP, not performed; E.h. Enterobacter hormaechei; E.b, Enterobacter bugandensis.NNNNYYYNNYNNm/z (calculated)Peaks present in the spectra1388.7+++++1599.1+1717.4++++1745.5+++++++++1769.3++++++1783.4+1797.4++++" @default.
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• W3187992502 title "Presence of 2-hydroxymyristate on endotoxins is associated with death in neonates with Enterobacter cloacae complex septic shock" @default.
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