Contaminated Incubators: Source of a Multispecies Enterobacter Outbreak of Neonatal Sepsis.

The genus Enterobacter includes species responsible for nosocomial outbreaks in fragile patients, especially in neonatal intensive care units (NICUs). Determining the primary source of infection is critical to outbreak management and patient outcomes. In this investigation, we report the management and control measures implemented during an Enterobacter outbreak of bloodstream infections in premature babies. The study was conducted in a French NICU over a 3-year period (2016 to 2018) and included 20 premature infants with bacteremia. The clinical and microbiological characteristics were identified, and whole-genome sequencing (WGS) was performed on bacteremia isolates. Initially, several outbreak containment strategies were carried out with no success. Next, outbreak investigation pinpointed the neonatal incubators as the primary reservoir and source of contamination in this outbreak. A new sampling methodology during "on" or "in use" conditions enabled its identification, which led to their replacement, thus resulting in the containment of the outbreak. WGS analysis showed a multiclonal outbreak. Some clones were identified in different isolation sources, including patients and neonatal incubators. In addition, microbiological results showed a multispecies outbreak with a high prevalence of Enterobacter bugandensis and Enterobacter xiangfangensis. We conclude that the NICU health care environment represents an important reservoir for Enterobacter transmission and infection. Finally, extracting samples from the neonatal incubator during active use conditions improves the recovery of bacteria from contaminated equipment. This method should be used more frequently to achieve better monitoring of the NICU for HAIs prevention. IMPORTANCE Neonatal incubators in the NICU can be an important reservoir of pathogens responsible for life-threatening outbreaks in neonatal patients. Traditional disinfection with antiseptics is not sufficient to eradicate the microorganisms that can persist for long periods in the different reservoirs. Identification and elimination of the reservoirs are crucial for outbreak prevention and control. In our investigation, using a new strategy of microbiological screening of neonatal incubators, we demonstrated that these were the primary source of contamination. After their replacement, the outbreak was controlled. This new methodology was effective in containing this outbreak and could be a viable alternative for infection prevention and control in outbreak situations involving incubators as a reservoir.

dnaJ: a New Approach to Identify Species within the Genus Enterobacter.

The taxonomy of the genus Enterobacter can be confusing and has been considerably revised in recent years. We propose a PCR and amplicon sequencing technique based on a partial sequence of the dnaJ gene for species assignment consistent with DNA-DNA digital hybridization (dDDH) and pairwise average nucleotide identity (ANI). We performed a validation of the method by comparing the type strains of each species, sequences obtained from the GenBank database, and clinical specimens. Our results show that the polymorphism of the target sequence of dnaJ allows the identification of species. Using this gene, we assigned the species to 100 strains deposited in the GenBank database that were consistent with the species assignment by dDDH and ANI. The analysis showed that using the partial dnaJ sequence is congruent with WGS as far as correct identification of Enterobacter species is concerned. Finally, we applied our dnaJ method on a national collection of 68 strains identified as Enterobacter isolated from the blood cultures of premature babies using an algorithm based on a type-strain library and the SeqScape software. For the first time, we identified Enterobacter quasihormaechei in blood cultures from four neonatal sepsis cases. We also noticed a higher prevalence of E. bugandensis (36.3%; 32/88) and E. xiangfangensis (46.5%; 41/88). E. bugandensis is a novel species recently described specifically in instances of neonatal sepsis. In conclusion, sequencing a part of the dnaJ gene could be a quick, more economical, and highly discriminating method of identifying Enterobacter species in clinical practice and research. IMPORTANCE We propose a new approach for Enterobacter species identification based on the diversity of the gene encoding the heat shock protein DnaJ. This new tool can be easily implemented in clinical laboratories in addition to identification by MALDI-TOF.

Presence of 2-hydroxymyristate on endotoxins is associated with death in neonates with Enterobacter cloacae complex septic shock.

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.

Structure activity characterization of Bordetella petrii lipid A, from environment to human isolates.

Bordetella petrii, a facultative anaerobic species, is the only known member of the Bordetella genus with environmental origin. However it was also recently isolated from humans. The structures of the B. petrii lipid A moieties of the endotoxins were characterized here for the first time for an environmental strain and compared to that of human isolates. Characterization was achieved using chemical analyses, gas chromatography-mass spectrometry, and Matrix Assisted Laser Desorption Ionisation mass spectrometry. The analyses revealed that the different lipid A structures contain a common bisphosphorylated ß-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide as well at the C-3' in ester linkages. Similar to Bordetella pertussis and Bordetella bronchiseptica lipids A, the hydroxytetradecanoic acid at the C-2' position was substituted by tetradecanoic acid. Unlike B. pertussis, the hydroxytetradecanoic acid at the C-2 position was substituted with either 12:0 or 14:0 and/or their 2-OH forms. Depending on the environmental or human origin the structures differed in the length and degree of fatty acid acylation and impacted the IL-6 and TNF-α inflammatory responses tested. In one isolate we showed the presence at the C-3 position of the short-chain 10:0(3-OH), which according to our previous analyses is more characteristic of the human pathogens in the genus like B. pertussis and Bordetella parapertussis.

Desulfovibrio desulfuricans isolates from the gut of a single individual: structural and biological lipid A characterization.

The levels of sulfate-reducing bacteria (SRB), including Desulfovibrionaceae, in the gut increase following a fat-enriched diet. Endotoxins from gut microbiota contribute to the inflammation process, leading to metabolic diseases. Thus, we sought to characterize the lipid A structures of Desulfovibrionaceae lipopolysaccharides (LPS) that are associated with the microbiota inflammatory properties. LPS variants were obtained from two SRB isolates from the gut of a single individual. These LPS variants shared similar lipid A moieties with Enterobacterial LPS, but differed from one another with regard to fatty-acid numbers and endotoxic activity. This first complete structural characterization of Desulfovibrio lipid A gives new insights into previously published data on Desulfovibrio lipid A biosynthesis. LPS microdiversity within SRBs illustrates how adaptation can influence pro-inflammatory potential.

Interactions between LPS and lung surfactant proteins.

After penetration into the lower airways, bacterial lipopolysaccharide (LPS) interacts with alveolar cells in a fluid environment consisting of pulmonary surfactant, a lipid-protein complex which prevents alveolar collapsing and participates in lung defense. The two hydrophilic surfactant components SP-A and SP-D are proteins with collagen-like and lectin domains (collectins) able to interact with carbohydrate-containing ligands present on microbial membranes, and with defined regions of LPS. This explains their capacity to damage the bacterial envelope and induce an antimicrobial effect. In addition, they modulate LPS-induced production of pro-inflammatory mediators in leukocytes by interaction with LPS or with leukocyte receptors. A third surfactant component, SP-C, is a small, highly hydrophobic lipopeptide which interacts with lipid A and reduces LPS-induced effects in macrophages and splenocyte cultures. The interaction of the different SPs with CD14 might explain their ability to modulate some LPS responses. Although the alveolar fluid contains other antiLPS and antimicrobial agents, SPs are the most abundant proteins which might contribute to protect the lung epithelium and reduce the incidence of LPS-induced lung injury. The presence of the surfactant collectins SP-A and SP-D in non-pulmonary tissues, such as the female genital tract, extends their field of action to other mucosal surfaces.

Cellular antiendotoxin activities of lung surfactant protein C in lipid vesicles.

The respiratory system is continuously exposed to airborne particles containing lipopolysaccharide. Our laboratory established previously that the hydrophobic surfactant protein C (SP-C) binds to lipopolysaccharide and to one of its cellular receptors, CD14. Here we examined the influence of SP-C, and of a synthetic analog, on some cellular in vitro effects of lipopolysaccharide. When associated with vesicles of dipalmitoylphosphatidylcholine, SP-C inhibits the binding of a tritium-labeled lipopolysaccharide to the macrophage cell line RAW 264.7. Under similar conditions of presentation, SP-C inhibits the mitogenic effect of lipopolysaccharide on mouse splenocytes, and inhibits the lipopolysaccharide-induced production of tumor necrosis factor-alpha by peritoneal and alveolar macrophages, and of nitric oxide by RAW 264.7 cells. In contrast, tumor necrosis factor-alpha production induced by a lipopeptide, and nitric oxide production induced by picolinic acid, were not affected by SP-C. The lipopolysaccharide-binding capacity of SP-C is resistant to peroxynitrite, a known mediator of acute lung injury formed by reaction of nitric oxide with superoxide anions. These results indicate that SP-C may play a role in lung defense; SP-C resists degradation under inflammatory conditions and traps lipopolysaccharide, preventing it from inducing production of noxious mediators in alveolar cells.

Histones: a novel class of lipopolysaccharide-binding molecules.

Unlike soluble and membrane forms of lipopolysaccharide (LPS)-binding proteins, intracellular LPS-binding molecules are poorly documented. We looked for such molecules in a murine lung epithelial cell line. Two proteins with LPS-binding activity were isolated and unambiguously identified as histones H2A.1 and H4 by mass spectrometry. Synthetic peptides representing partial structures indicated that the LPS binding site is located in the C-terminal moiety of the histones. Extending the study, we found that histones H1, H2A, H2B, H3, and H4 from calf thymus are all able to bind LPS. Bindings were specific, and affinities, determined by isothermal titration calorimetry, were (except for H4) higher than that of the LPS-binding antibiotic polymyxin B. In the presence of H2A the binding of LPS to the macrophage cell line RAW 264.7, and the LPS-induced production of TNF-alpha and nitric oxide by these cells, were markedly reduced. Histones may thus represent a new class of intracellular and extracellular LPS sensors.

Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide.

In addition to their effects on alveolar surface tension, some components of lung surfactant also have immunological functions. We found recently that the hydrophobic lung surfactant protein SP-C specifically binds to the lipid A region of lipopolysaccharide (LPS). In this study, we show that SP-C also interacts with CD14. Four observations showed cross talk between the three molecules SP-C, LPS, and CD14. (i) Like LBP, SP-C allows the binding of a fluorescent LPS to cells expressing CD14 (the other surfactant components were ineffective). (ii) Recombinant radiolabeled CD14 and SP-C (or a synthetic analog of SP-C) interact in a dose-dependent manner. (iii) LPS blocks the binding of radiolabeled CD14 to SP-C-coated wells. (iv) SP-C enhances the binding of radiolabeled CD14 to LPS-coated wells. These results, obtained with native murine SP-C and with three synthetic analogs, suggest that LPS and CD14 interact with the same region of SP-C and that binding of SP-C modifies the conformation of CD14 or the accessibility of its LPS-binding site, allowing it to bind LPS. This ability of SP-C to interact with the pattern recognition molecule CD14 extends the possible immunological targets of SP-C to a large panel of microorganisms that can enter the airways.

Structural basis for interactions between lung surfactant protein C and bacterial lipopolysaccharide.

In the respiratory tract, recognition of bacterial endotoxin (lipopolysacharide, LPS) is a critical step of the innate host defense system directed against invading pathogens. Secretions of the airways contain proteins that have direct antimicrobial activity (lysozyme, lactoferrin, defensins, and cathelicidins) as well as complement factors and surfactant proteins that contribute to host defense. The hydrophobic surfactant protein C (SP-C) recognizes LPS (Augusto, L., Le Blay, K., Auger, G., Blanot, D., and Chaby, R. (2001) Am. J. Physiol. 281, L776-L785). In the present study, using synthetic analogs of SP-C, we demonstrate that the palmitoyl residues of SP-C are not required for the interaction with LPS and that both the hydrophilic and hydrophobic regions of SP-C are required for specific binding of a radiolabeled rough-type LPS. In addition, using LPS submitted to different chemical treatments as well as synthetic analogs of the lipid A moiety of LPS, we established that the terminal phosphate group at the reducing end of the lipid A disaccharide in alpha configuration is of crucial importance for recognition by SP-C. The N-linked fatty acyl chain on the reducing glucosamine of lipid A also takes part in the interaction. Dipalmitoyl phosphatidylcholine is not specifically required for the LPS-binding activity of SP-C, although a lipid environment significantly increases the binding. These results provide a basis for experiments on the role of SP-C in presentation of LPS to alveolar cells and for the design of drugs for the management of endotoxin-induced lung injury.