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Meningitis-Associated Escherichia coli
Abstract
Meningitis-Associated Escherichia coli, Page 1 of 2
Abstract
Escherichia coliis the most common Gram-negative organism causing neonatal meningitis. Neonatal E. colimeningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis and pathophysiology contributes to such mortality and morbidity. Recent reports of neonatal meningitis caused by E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. E. colipenetration into the brain, the essential step in the development of E. coli meningitis, requires a high-degree of bacteremia and penetration of the blood-brain barrier as live bacteria, but the underlying mechanisms remain incompletely understood. Recent functional genomic approaches of meningitis-causing E. coli in both in vitro and in vivo models of the blood-brain barrier (e.g., human brain microvascular endothelial cells and animal models of experimental hematogenousE. colimeningitis, respectively) have identified several E. coli factors contributing to a high-degree of bacteremia, as well as specific microbial factors contributing to E. coli invasion of the blood-brain barrier. In addition, E. coli penetration of the blood-brain barrier involves specific host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors and host cell signaling molecules is efficient in preventing E. coli penetration into the brain. Continued investigation of the microbial and host factors contributing to E. colibacteremia andinvasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis, thereby limiting the exposure to emerging antimicrobial-resistant E. coli.
... Predictive functional analysis of bacterial communities showed that the main functions of the bacteria in 4 groups were distinct from each other. Bacterial motility is beneficial for the survival of bacteria (Halverson, 2005) and has been shown to contribute to the virulence of many enteric bacteria (Kim, 2013), particularly those infecting the mucosal surface (Flo and Aderem, 2005), with harmful effects on host health. Hence, the higher abundance of bacteria shown to be highly relevant to motility and protein repair in the NC group indicated a worse intestinal microbial environment, which accounted for the highest fecal score in this group. ...
This study was conducted to investigate the effects of spray-dried porcine plasma protein (SDPP) or spray-dried chicken plasma protein (SDCP) supplementation in diets without the inclusion of antibiotics and zinc oxide (ZnO) on growth performance, fecal score, and fecal microbiota in early-weaned piglets. A total of 192 healthy weaning piglets (Duroc × Landrace × Yorkshire, 21 d old) were blocked by BW (6.53 ± 0.60 kg) and randomly assigned to 4 dietary treatments: negative control (NC, basal diet), positive control (PC), basal diet + ZnO at 2 g/kg and antibiotics at 0.8 g/kg), SDPP (containing 5% SDPP), and SDCP (containing 5% SDCP). The experiment lasted 14 d. The SDPP group had higher (P < 0.05) final BW, average daily gain and average daily feed intake than the NC and SDCP groups. The percentage of piglets with fecal scores at 2 or ≥ 2 was higher (P < 0.05) in the NC and SDCP groups than in the PC group. A decreased (P < 0.05) bacterial alpha diversity and Bacteroidetes abundance, but increased (P < 0.05) Firmicutes abundance were observed in the PC and SDPP groups when compared to the NC group. The relative abundance of Lactobacillus was higher (P < 0.05) in the SDPP than in the SDCP group, and that of Streptococcus was higher (P < 0.01) in the PC and SDPP groups than in the NC group. The PC group also had higher (P < 0.01) Faecalibacterium abundance than the NC and SDCP groups. Additionally, the SDCP group had higher (P < 0.05) serum urea nitrogen than those fed other diets, and lower (P < 0.10) short-chain fatty acids to branched-chain fatty acids ratio than the PC and SDPP groups. Overall, SDPP was a promising animal protein for piglets in increasing feed intake, modifying gut microbiota profile, reducing gut protein fermentation and alleviating diarrhea frequency, thus promoting growth performance, under the conditions with limited in-feed utilization of antibiotics and ZnO.
The proteome of two newborn meningitis Escherichia coli K1 (NMEC) morphotypes was examined via a label-free proteomics approach. Besides shared NMEC virulence factors, the two strains have different evolutionary strategies—strain IHE3034 tends to perform anaerobic respiration continuously, while strain RS218 maintains its filamentous morphotype due to active SOS response.
The species Escherichia coli represents an unfathomable variety of commensal, pathogenic, and environmental strains. The conventional cyclic di-GMP signaling in E. coli controls sessility-motility changes linked to commensalism and/or pathogenicity. Extraintestinal Pathogenic E. coli (ExPEC) are “commensals” that can cause an array of infections outside the gastrointestinal tract. To accommodate their pathogenic lifestyle with the commensal one, ExPEC biology is shaped not only by the presence of specific virulence genes and pathoadaptive mutations but also by regulatory adaptations. Bioinformatic and genetic studies indicate that the cyclic di-GMP signaling network is included in the adaptation process. For example, some neuroinvasive ExPEC were found to maintain reduced cyclic di-GMP levels due to RpoS deactivation, resulting in loss of appearance of the rugose morphotype. Moreover, ExPEC has a diversified repertoire of cyclic di-GMP degrading enzymes obtained by acquisition of novel genes often associated with fimbrial biogenesis gene clusters (e.g., sfaY/papY/focY) and by modification or deletion of specific core genome genes. For example, the majority of ExPEC contains a shortened allelic variant of the ycgG gene and some ExPEC strains do not even carry the genetic locus. New combinations of regulators offer a new cyclic di-GMP platform for S-fimbrial biogenesis and for new metabolic capabilities leading to citrate utilization and ferric citrate uptake. In this review, we outline the prerequisites for the unconventional signaling network, the rationale behind its existence in ExPEC, and future perspectives in studies of ExPEC.
Neonatal meningitis Escherichia coli (NMEC) is one of the top causes of neonatal meningitis worldwide. Here, 85 NMEC and 204 fecal E. coli isolates from healthy humans (HFEC) were compared for possession of traits related to virulence, antimicrobial resistance,
and plasmid content. This comparison was done to identify traits that typify NMEC and distinguish it from commensal strains
to refine the definition of the NMEC subpathotype, identify traits that might contribute to NMEC pathogenesis, and facilitate
choices of NMEC strains for future study. A large number of E. coli strains from both groups were untypeable, with the most common serogroups occurring among NMEC being O18, followed by O83,
O7, O12, and O1. NMEC strains were more likely than HFEC strains to be assigned to the B2 phylogenetic group. Few NMEC or
HFEC strains were resistant to antimicrobials. Genes that best discriminated between NMEC and HFEC strains and that were present
in more than 50% of NMEC isolates were mainly from extraintestinal pathogenic E. coli genomic and plasmid pathogenicity islands. Several of these defining traits had not previously been associated with NMEC
pathogenesis, are of unknown function, and are plasmid located. Several genes that had been previously associated with NMEC
virulence did not dominate among the NMEC isolates. These data suggest that there is much about NMEC virulence that is unknown
and that there are pitfalls to studying single NMEC isolates to represent the entire subpathotype.
Neonatal meningitis Escherichia coli (NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a
result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain,
the essential step for the development of E. coli meningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the
bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells,
the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that
NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement
regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI
may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with
C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this
bacterial factor a potential target for prevention and therapy of E. coli meningitis.
Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions
involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner.
Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed
that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was
examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response
to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association
with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC.
Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. Based on sequence analysis,
we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain
microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including
Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ
in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and
induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings
demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release
followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing
E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.
Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to contribute to invasion by meningitis-causing Escherichia coli K1 of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. However, CNF1 is a cytosolic protein and it remains unclear how its secretion occurs, contributing to E. coli invasion of HBMEC. To investigate the genetic requirement for CNF1 secretion in E. coli K1 strain RS218, we performed mini-Tn5 in vitro mutagenesis and constructed a transposon mutant library of strain NBC, in which β-lactamase was fused to the C-terminus of CNF1 in the chromosome of strain RS218. We identified a transposon mutant (NBC-1E6) that exhibited reduced β-lactamase activity in its culture supernatant and had the transposon inserted into the ygfZ gene. When ygfZ was deleted from the genome of strain RS218 (ΔygfZ), the translocation of CNF1 into HBMEC was impaired. Subcellular localization analysis of CNF1 demonstrated that YgfZ, a periplasmic protein, contributes to secretion of CNF1 into outer-membrane vesicles (OMVs). The ΔygfZ mutant was significantly defective in invasion of HBMEC compared to the parent E. coli K1 strain. The defects of the ΔygfZ mutant in CNF1 secretion into OMVs and translocation into HBMEC as well as invasion of HBMEC were abrogated by complementation with ygfZ. Taken together, our findings demonstrate that YgfZ contributes to CNF1 secretion into OMVs in meningitis-causing E. coli K1.
Neonatal bacterial meningitis continues to be an important cause of mortality and morbidity worldwide. Escherichia coli possessing the K1 capsular polysaccharide is the most common Gram-negative pathogen causing neonatal meningitis. Here we
present the complete genome sequence of neonatal meningitis-associated E. coli strain CE10, a unique K1 strain with a functional type III secretion system. Functional analysis of the genome should enhance
our knowledge of the pathogenesis of neonatal E. coli K1 meningitis.
Alpha7 nicotinic acetylcholine receptor (nAChR), an essential regulator of inflammation, is abundantly expressed in hippocampal neurons, which are vulnerable to bacterial meningitis. However, it is unknown whether α7 nAChR contributes to the regulation of these events. In this report, an aggravating role of α7 nAChR in host defense against meningitic E. coli infection was demonstrated by using α7-deficient (α7(-/-)) mouse brain microvascular endothelial cells (BMEC) and animal model systems. As shown in our in vitro and in vivo studies, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the blood-brain barrier (BBB) were significantly reduced in α7(-/-) BMEC and α7(-/-) mice. Stimulation by nicotine was abolished in the α7(-/-) cells and animals. The same blocking effect was achieved by methyllycaconitine (α7 antagonist). The tight junction molecules occludin and ZO-1 were significantly reduced in the brain cortex of wildtype mice infected with E. coli and treated with nicotine, compared to α7(-/-) cells and animals. Decreased neuronal injury in the hippocampal dentate gyrus was observed in α7(-/-) mice with meningitis. Proinflammatory cytokines (IL-1β, IL-6, TNFα, MCP-1, MIP-1alpha, and RANTES) and adhesion molecules (CD44 and ICAM-1) were significantly reduced in the cerebrospinal fluids of the α7(-/-) mice with E. coli meningitis. Furthermore, α7 nAChR is the major calcium channel for nicotine- and E. coli K1-increased intracellular calcium concentrations of mouse BMEC. Taken together, our data suggest that α7 nAChR plays a detrimental role in the host defense against meningitic infection by modulation of pathogen invasion, PMN recruitment, calcium signaling and neuronal inflammation.
Escherichia coli is the most common Gram-negative organism causing neonatal meningitis. Previous studies demonstrated that E. coli K1 invasion of brain microvascular endothelial cells (BMEC) is required for penetration into the central nervous system,
but the microbe-host interactions that are involved in this process remain incompletely understood. Here we report the involvement
of vascular endothelial growth factor receptor 1 (VEGFR1) expressed on human brain microvascular endothelial cells (HBMEC)
in E. coli K1 invasion of HBMEC. Our results showed that treatment of confluent HBMEC with pan-VEGFR inhibitors significantly inhibited
E. coli K1 invasion of HBMEC. Immunofluorescence results indicated the colocalization of VEGFR1 with E. coli K1 during bacterial invasion of HBMEC. The E. coli-induced actin cytoskeleton rearrangements in HBMEC were blocked by VEGFR inhibitors but not by VEGFR2-specific inhibitors.
The small interfering RNA (siRNA) knockdown of VEGFR1 in HBMEC significantly attenuated E. coli invasion and the concomitant actin filament rearrangement. Furthermore, we found an increased association of VEGFR1 with
the p85 subunit of phosphatidylinositol 3-kinase (PI3K) in HBMEC infected with E. coli K1 and that E. coli K1-triggered Akt activation in HBMEC was blocked by VEGFR1 siRNA and VEGFR inhibitors. Taken together, our results demonstrate
that VEGFR1 contributes to E. coli K1 invasion of HBMEC via recruitment of the PI3K/Akt signaling pathway.
Escherichia coli K1 meningitis occurs following penetration of the blood-brain barrier, but the underlying mechanisms involved in E. coli penetration of the blood-brain barrier remain incompletely understood. We have previously shown that host cytosolic phospholipase
A2α (cPLA2α) contributes to E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying
mechanisms remain unclear. cPLA2α selectively liberates arachidonic acid from membrane phospholipids. Here, we provide the first direct evidence that host
5-lipoxygenase and lipoxygenase products of arachidonic acid, cysteinyl leukotrienes (LTs), contribute to E. coli K1 invasion of HBMEC and penetration into the brain, and their contributions involve protein kinase C alpha (PKCα). These
findings demonstrate that arachidonic acid metabolism regulates E. coli penetration of the blood-brain barrier, and studies are needed to further elucidate the mechanisms involved with metabolic
products of arachidonic acid for their contribution to E. coli invasion of the blood-brain barrier.
Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding
of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling
molecules including cytosolic phospholipase A2α (cPLA2α). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited
E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released
by cPLA2α, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide
and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC
receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.
Ineffectiveness of antibiotics in treating neonatal Escherichia coli K1 meningitis and the emergence of antibiotic-resistant strains evidently warrants new prevention strategies. We observed that administration of interleukin (IL)-10 during high-grade bacteremia clears antibiotic-sensitive and -resistant E. coli from blood of infected mice. Micro-CT studies of brains from infected animals displayed gross morphological changes similar to those observed in infected human neonates. In mice, IL-10, but not antibiotic or anti-TNF antibody treatment prevented brain damage caused by E. coli. IL-10 administration elevated CR3 expression in neutrophils and macrophages of infected mice, whereas infected and untreated mice displayed increased expression of FcgammaRI and TLR2. Neutrophils or macrophages pretreated with IL-10 ex vivo exhibited a significantly greater microbicidal activity against E. coli compared with cells isolated from wild-type or IL-10-/- mice. The protective effect of IL-10 was abrogated when CR3 was knocked-down in vivo by siRNA. The increased expression of CR3 in phagocytes was caused by inhibition of prostaglandin E-2 (PGE-2) levels, which were significantly increased in neutrophils and macrophages upon E. coli infection. These findings describe a novel modality of IL-10-mediated E. coli clearance by diverting the entry of bacteria via CR3 and preventing PGE-2 formation in neonatal meningitis.
Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of
the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial
determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion
was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42°C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation
on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored
protein in E. coli, and the nlpI mutant was defective in cytosolic phospholipase A2α (cPLA2α) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpI's contribution
to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA2α.
We previously showed that cytotoxic necrotizing factor 1 (CNF1) contributes to Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) and interacts with the receptor on the surface of HBMEC.
CNF1 is the cytoplasmic protein, and it remains incompletely understood how CNF1 is secreted across the inner and outer membranes
in E. coli K1. In order to investigate the genetic determinants for secretion of CNF1 in E. coli K1, we performed Tn5 mutagenesis screening by applying β-lactamase as a reporter to monitor secretion of CNF1. We identified a Tn5 mutant that exhibited no β-lactamase activity in the culture supernatant and in which the mutated gene encodes a ferredoxin
gene (fdx). In the fdx deletion mutant, there was no evidence of translocation of CNF1 into HBMEC. Western blot analysis of the fdx deletion mutant revealed that ferredoxin is involved in translocation of CNF1 across the cytoplasmic membrane. The fdx mutant exhibited significantly decreased invasion of HBMEC, similar to the decreased HBMEC invasion observed with the CNF1
mutant. The failures to secrete CNF1 and invade HBMEC of the fdx mutant were restored to the levels of the parent strain by complementation with fdx. These findings demonstrate for the first time that ferredoxin is involved in secretion of CNF1 across the inner membrane
in meningitis-causing E. coli K1.
Although Escherichia coli strains possessing the K1 capsule are predominant among isolates from neonatal E. coli meningitis and most of these K1 isolates are associated with a limited number of 0 lipopolysaccharide (LPS) types, the basis of this association of K1 and certain 0 antigens with neonatal E. coli meningitis is not clear. The present study examined in experimental E. coli bacteremia and meningitis in newborn and adult rats whether or not the K1 capsule and/or O-LPS antigen are critical determinants in the development of meningitis. Rats received subcutaneously at K1 E. coli strain (018+K1+) or mutants lacking either the K1 capsule (018+K1-) or 0 side-chain (018-K1+). 12-24 h later, blood and cerebrospinal fluid (CSF) specimens were obtained for quantitative cultures. The isolation of E. coli from CSF was observed in both newborn and adult rats infected with K1+ strains regardless of LPS phenotype (018+ or 18-) who also developed a high degree of bacteremia (e.g., greater than 10(4) CFU/ml of blood). In contrast, none of the newborn and adult rats infected with 018+K1- and developing bacteremia of greater than 10(4) were found to have positive CSF cultures. These findings indicate that the presence of the K1 capsule and a high degree of bacteremia are key determinants in the development of E. coli meningitis, suggesting that there may be specific binding sites present in the brain which have an affinity for the K1 capsule and thus may be responsible for the entry of K1-encapsulated E. coli into the meninges.
Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal meningitis, we have studied the presence of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat organs and their binding was assessed by indirect immunofluorescence. In the brain of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and brain ventricles. The binding was completely inhibited by the trisaccharide NeuAc alpha 2-3Gal beta 1-4Glc, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressing S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, whereas the non-fimbriated host strain and a recombinant strain expressing P fimbriae did not adhere to brain tissues. The results suggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatal brain has a pathogenetic role during bacterial invasion from circulation into the cerebrospinal fluid.
In an attempt to further assess the role of S-fimbriae in the pathogenesis of Escherichia coli meningitis, the adherence of E. coli strains with or without S-fimbriae were examined for this study to purified glycolipids using thin layer chromatography overlay assays. Only S-fimbriated E. coli strains bound to sulfatide, seminolipid, galactosyl ceramide, and lactosyl ceramide but not to gangliosides including sialyl neolacto-series and other neutral glycolipids. The binding of S-fimbriated E. coli to sulfatide was temperature dependent (i.e. maximal at 37 degrees C) and inhibited by S-fimbriae, anti-S-fimbriae, and anti-S-adhesin antibodies as well as by sulfatide, galactosyl ceramide, and lactosyl ceramide. E. coli transformants which lack the sfaA gene from the Sfa gene cluster showed no binding to the glycolipids, while other transformants lacking the adhesin gene sfaS or sfaG or H and mutants obtained by site-directed mutagenesis in the sfaS gene exhibited a similar binding to the glycolipids compared to the parent S-fimbriated strain. A large amount of sulfated glycolipids was demonstrated on brain endothelial cells and the binding of S-fimbriated E. coli to brain endothelial cells was inhibited by these glycolipids. These findings suggest that the binding of S-fimbriated E. coli to brain endothelial cells occurs in part via glycolipids containing terminal Gal(3SO4)beta-1 residues and in part by S-fimbriae protein SfaA. S-adhesin was not involved in the binding of S-fimbriae to these glycolipids.
The laminin-based nonapeptide Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR) and pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR) have been previously demonstrated to support the attachment of several cell types and to competitively bind to the 67-kDa high affinity laminin receptor. Cell attachment, but not spreading, on substrates containing adsorbed CDPGYIGSR or YIGSR was observed. In this report we describe YIGSR-mediated attachment and spreading of a wide variety of cell types. GYIGSRY promoted cell spreading and stress fiber formation when it was covalently immobilized through the amino-terminal Gly residue, used as a spacer arm. Spreading was not observed when adsorbed YIGSR peptide was used. Functionally blocking antiserum directed against the 67-kDa and related laminin-binding proteins blocked human foreskin fibroblast (HFF) spreading, but not attachment, on covalently grafted GYIGSRY substrates. However, functionally blocking antisera directed against the vitronectin receptor, integrin alpha v beta 3, and the fibronectin receptor, integrin alpha 5 beta 1, did not affect HFF spreading on these substrates. When HFFs spread on these substrates, the 67-kDa laminin receptor co-localized with the cytoplasmic proteins alpha-actinin and vinculin into discrete structures. These results suggest that the adhesion ligand YIGSR is solely sufficient for cell spreading when it is conformationally constrained by covalent attachment to a solid substrate, at least when attached via its amino terminus. Furthermore, the role of the 67-kDa laminin receptor in recognition of this ligand and mediating cell attachment is confirmed in this study. This report also provides the first evidence for direct or indirect association of this receptor with vinculin and alpha-actinin when YIGSR-mediated cell spreading occurs.
The structurally similar polysialic acid capsules of group B meningococci and Escherichia coli K1 are poor immunogens, and attempts are currently being made to improve their immunogenicity by chemical modifications. An IgG monoclonal antibody to these polysialic acid capsules was used for the study of the presence of structurally similar components in tissue glycoproteins to investigate the reasons for the poor immunogenicity and to evaluate potential dangers in active or passive immunization. By immunoblotting polysialic acid was detected outside the brain in newborn rat kidney, heart, and muscle. It appeared in immunoblots as one component and with similar mobility to the neural cell adhesion molecule N-CAM. Specificity studies of the antibody and endosialidase treatment showed that the polysialic acid glycans detected were composed of chains as long as eight sialic acid residues or more. The polysialic acid was not detected in the corresponding tissues of the adult animal. These results indicate that polysialic acid units are developmentally regulated components of both neural and extraneural tissues, and are bound to components with properties similar to a known cell-adhesion molecule. This together with the presence of low amounts of polysialic acid even in the adult brain, suggests potential hazards in vaccination trials and suggested immunotherapy of meningitis caused by group B meningococci or E. coli K1, which should be carefully assessed.
Group B streptococci (GBS) are the leading cause of meningitis in newborns. Although meningitis develops following bacteremia, the precise mechanism or mechanisms whereby GBS leave the bloodstream and gain access to the central nervous system (CNS) are not known. We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells (BMEC), the single-cell layer which constitutes the blood-brain barrier. In order to test this hypothesis, we developed an in vitro model with BMEC isolated from a human, immortalized by simian virus 40 transformation, and propagated in tissue culture monolayers. GBS invasion of BMEC monolayers was demonstrated by electron microscopy. Intracellular GBS were found within membrane-bound vacuoles, suggesting the organism induced its own endocytic uptake. GBS invasion of BMEC was quantified with a gentamicin protection assay. Serotype III strains, which account for the majority of CNS isolates, invaded BMEC more efficiently than strains from other common GBS serotypes. GBS survived within BMEC for up to 20 h without significant intracellular replication. GBS invasion of BMEC required active bacterial DNA, RNA, and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton. The polysaccharide capsule of GBS attenuated the invasive ability of the organism. At high bacterial densities, GBS invasion of BMEC was accompanied by evidence of cellular injury; this cytotoxicity was correlated to beta-hemolysin production by the bacterium. Finally, GBS demonstrated transcytosis across intact, polar BMEC monolayers grown on Transwell membranes. GBS invasion of BMEC may be a primary step in the pathogenesis of meningitis, allowing bacteria access to the CNS by transcytosis or by injury and disruption of the endothelial blood-brain barrier.
To identify critical microbial determinant(s) in serum resistance and in vivo virulence, 5 strains of Escherichia coli with different combinations of K and O types were examined along with their unencapsulated mutants. All 3 E. coli strains possessing the K1 capsule were considerably more resistant to normal human serum in vitro and more virulent in newborn rats than their isogenic mutants lacking the K1 capsule. In contrast, two K5 encapsulated strains and their unencapsulated mutants were essentially the same for their degree of serum resistance and in vivo virulence. These findings suggest that for the demonstration of serum resistance and in vivo virulence, a capsule is required for E. coli strains encapsulated with K1, whereas certain E. coli strains encapsulated with K5 may not need a capsule.
One hundred and thirty-one strains of Escherichia coli isolated from the cerebrospinal fluid of patients in the United Kingdom were tested for resistance to 13 antimicrobial drugs. Sixty-four strains (49%) were resistant to one or more drugs and 44 (34%) were resistant to three or more drugs. Resistance to ampicillin, sulphonamides, streptomycin and tetracycline occurred most frequently.
Most cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood–brain barrier. We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E. coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo. In this study, differential fluorescence induction (DFI) was used to identify E. coli K1 genes involved in this differentially expressed invasion phenotype. E. coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions. Twenty-four clones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e. NBS). Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion-repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus). Among the other NBS-inducing loci, finPtraJ was identified as well as several clones with no homology to other known genes. When ygdP, finPtraJ and several of the unique loci were disrupted in E. coli K1, there was a significant decrease in human BMEC (HBMEC) invasion. RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.
In an attempt to examine whether routes of bacterial entry into the central nervous system have any bearing on subsequent changes in blood–brain barrier permeability, we examined cerebrospinal fluid (CSF) penetration of circulating125I-albumin in two different models of experimental meningitis due to K1Escherichia coli,type III group B streptococcus, orHaemophilus influenzaetype b in infant rats: hematogenous meningitis subsequent to subcutaneous inoculation of bacteria vs meningitis induced by direct inoculation of bacteria into the CSF via the cisterna magna. In the model of hematogenous meningitis, the mean CSF penetration was significantly greater in animals withH. influenzaetype b meningitis than in those with meningitis due to K1E. colior type III group B streptococcus. In contrast, the mean CSF penetration was significantly enhanced in all animals with meningitis induced by intracisternal inoculation regardless of infecting pathogens. Tumor necrosis factor activity in CSF appeared to correlate with the functional penetration of circulating albumin across the blood–brain barrier in both models of experimental meningitis. These findings suggest that the alterations of blood–brain barrier permeability during development of experimental meningitis may vary for different models of inducing meningitis and that the mechanisms responsible for these different permeability changes may be multifactorial.
Neonatal Escherichia coli meningitis continues to be an important cause of mortality and morbidity throughout the world. The major contributing factors to this mortality and morbidity include our incomplete knowledge on its pathogenesis and an emergence of antimicrobial-resistant E. coli. Recent reports of neonatal meningitis caused by E. coli producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge, and innovative approaches are needed to identify potential targets for prevention and therapy of E. coli meningitis.
E. coli invasion of the blood-brain barrier is a prerequisite for penetration into the brain and requires specific microbial-host factors as well as microbe-specific and host-specific signaling molecules. Recent studies identified additional microbial and host factors contributing to E. coli invasion of the blood-brain barrier and elucidated their underlying mechanisms. Blockade of the microbial-host factors contributing to E. coli invasion of the blood-brain barrier was shown to be efficient in preventing E. coli penetration into the brain.
Continued investigation on the microbial-host factors contributing to E. coli invasion of the blood-brain barrier is needed to identify new targets for prevention and therapy of E. coli meningitis, thereby limiting the exposure to emerging antimicrobial-resistant E. coli.
Infections due to extraintestinal pathogenic E. coli (ExPEC) are very common in humans as well as in animals. In humans ExPEC infections include urinary tract infections (UTI), septicemia, and wound infections, which result in significant morbidity, mortality, and substantial healthcare costs. In view of the increasing number of ExPEC infections caused by more and more resistant strains, effective prevention would be desirable. Given the rising treatment costs, a vaccine may be cost-effective in selected patient groups, such as women with recurrent UTI, patients with neurologic disorders impairing bladder function and men with prostate hyperplasia. Previous vaccine studies used single target proteins or whole inactivated ExPEC cells. Here, we describe a vaccine system for oral application based on artificial multiple subunit vaccine proteins. Those multi-epitope proteins are composed of predicted epitopes derived from ExPEC virulence-associated proteins. As ExPEC are known to form intracellular biofilms in the urothelium and can also resist killing by non-activated macrophages, T-cell responses are supposed to be an important measure to counteract these stages of ExPEC during infection. Therefore, a live bacterial antigen delivery system based upon the Salmonella type-III secretion system (T3SS) was used in this study to directly deliver the vaccine proteins into the cytoplasm of the host cells. Epitope-rich domains of the proteins FyuA, IroN, ChuA, IreA, Iha, and Usp were expressed in an attenuated Salmonella enterica serovar Typhimurium strain and translocated into target cells for extended periods of time inducing a strong T-cell response. No significant antibody titre increase against the secreted vaccine proteins could be detected in vaginal wash or serum. Despite that, one of the vaccine proteins was able to significantly reduce bacterial load in the challenge model of intraperitoneal sepsis. This study shows that a vaccine encompassing distinct epitopes of virulence-associated ExPEC proteins (i) can be applied for a T3SS-dependent vaccination strategy, (ii) elicits T-cell responses and (iii) confers protection after a single application.
Bacterial meningitis causes persisting neurofunctional sequelae. Theoccurrence of apoptotic cell death in the hippocampal subgranular zone of the dentate gyrus characterizes the disease in patients and relates to deficits in learning and memory in corresponding experimental models. Here, we investigated why neurogenesis fails to regenerate the damage in the hippocampus associated with the persistence of neurofunctional deficits. In an infant rat model of bacterial meningitis, the capacity of hippocampal-derived cells to multiply and form neurospheres was significantly impaired comparedto that in uninfected littermates. In an in vitro model of differentiating hippocampal cells, challenges characteristic of bacterial meningitis (i.e. bacterial components, tumor necrosis factor [20 ng/mL], or growth factor deprivation) caused significantly more apoptosis in stem/progenitor cells and immature neurons than in mature neurons. These results demonstrate that bacterial meningitis injures hippocampal stem and progenitor cells, a finding that may explain the persistence of neurofunctional deficits after bacterial meningitis.
Adjunctive therapies that reduce the cerebral edema in bacterial meningitis include osmotic agents. There is a lack of information comparing mannitol vs. hypertonic saline as an osmotic agent for adjunctive therapy of bacterial meningitis. We attempted to elucidate the impact of hypertonic saline in cerebral edema in the setting of bacterial meningitis as well as to explore potential mechanisms of action.
Randomized controlled in vivo study.
University research laboratory.
Rabbits.
A rabbit model of bacterial meningitis was used comparing 3% hypertonic saline with 20% mannitol as adjunctive therapy.
Adjunctive 3% hypertonic saline treatment persistently elevated mean arterial pressure as compared with the model or ampicillin group (p < .01). Although both 20% mannitol and 3% hypertonic saline efficiently elevated serum osmolality for almost 5 hrs (p < .01), 20% mannitol lowered intracranial pressure for only a short time (<2 hrs) and did not elevate cerebral perfusion pressure. Three percent hypertonic saline treatment efficiently lowered intracranial pressure and elevated cerebral perfusion pressure for almost 5 hrs (p < .01). Furthermore, 3% hypertonic saline treatment efficiently elevated serum Na+ concentration for >5 hrs (p < .01). Three percent hypertonic saline treatment was superior to 20% mannitol in lowering leukocyte number and protein content in cerebrospinal fluid (p < .01). Three percent hypertonic saline treatment reduced water content and Evans blue incorporation in the brain (p < .01). Three percent hypertonic saline treatment inhibited aquaporin 4 expression (p < .01) and attenuated pathologic brain damage more efficiently compared with adjuvant 20% mannitol treatment (p < .01).
Adjunctive 3% hypertonic saline treatment significantly elevated mean arterial pressure, reduced intracranial pressure, greatly improved cerebral perfusion pressure, inhibited brain aquaporin 4 expression, reduced cerebral edema, and attenuated brain damage with a superior effect over 20% mannitol in a rabbit bacterial meningitis model.
Neonatal meningitis by Eschericia coli RS218 occurs due to bacteremia and its transmigration across the blood-brain barrier. Although the outer membrane protein
A (OmpA), a molecule with extracellular loops has been shown to contribute to the above phenomenon, we do not know the exact
the role of these individual loops. Using bacterial strains whose individual loops have been removed, we demonstrated that
whereas Loops1 and 2 contribute to 70%–80% bacterial survival in serum, bacterial entry into human brain microvascular endothelial
cells (HBMEC) is governed by Loops1, 2, and 3. Cellular invasion was shown to require activation of host cytosolic phospholipase
A2 (cPLA2α) by Loops1 and 2 but not 3. This suggests 2 distinct pathways for bacterial entry into host cells. Loop 4 played
no role in either serum survival, cellular entry, or cPLA2α signaling. These findings demonstrate for the first time the different
contributions of extracellular loops of OmpA to the pathogenesis of E. coli meningitis.
Neurogenesis is increased in experimental models of bacterial meningitis. In this study, neurogenesis was examined after bacterial infection of the CNS, and after stroke and brain trauma in humans.
Brain sections of patients after death from bacterial meningitis, stroke, or brain trauma and from autopsy cases after death from nonneurologic diseases were investigated by immunohistochemistry.
In the dentate gyrus, the density of proliferating cellular nuclear antigen-expressing cells was higher after bacterial meningitis compared to the control group (p = 0.0075). Furthermore, the number of cells expressing the immature neuronal marker proteins TUC-4 and doublecortin were increased in brain sections of patients after death from meningitis compared to control cases (p = 0.0067 and p = 0.045). After stroke and brain trauma, higher densities of proliferating cells were observed (p = 0.031 and p = 0.018), while an increase of TUC-4-expressing cells was detected after stroke only (p = 0.0012 and p = 0.47).
The increased proliferation of neural progenitors suggests an endogenous mechanism in response to noxious stimuli. Stimulation of neurogenesis might help to alleviate the consequences of neuronal destruction in bacterial meningitis and other diseases of the brain.
Type III secretion systems (T3SSs) have been documented in many Gram-negative bacteria, including enterohemorrhagic Escherichia coli. We have previously shown the existence of a putative T3SS in meningitis-causing E. coli K1 strains, referred to as E. coli type III secretion 2 (ETT2). The sequence of ETT2 in meningitis-causing E. coli K1 strain EC10 (O7:K1) revealed that ETT2 comprises the epr, epa and eiv genes, but bears mutations, deletions and insertions. We constructed the EC10 mutants deleted of ETT2 or eivA gene, and their contributions to bacterial pathogenesis were evaluated in human brain microvascular endothelial cells (HBMECs). The deletion mutant of ETT2 exhibited defects in invasion and intracellular survival compared with the parental E. coli K1 strain EC10. The mutant deleted of eivA within ETT2 was also significantly defective in invasion and intracellular survival in HBMECs, and the defects of the eiv mutant were restored to the levels of the parent strain EC10 by transcomplementation. These findings suggest that ETT2 plays a role in the pathogenesis of E. coli K1 infection, including meningitis.
Although at least 100 different Escherichia coli capsular antigens have been recognised, strains possessing the K1 antigen are responsible for 77% of neonatal E. coli meningitis cases. K1 strains were found in 20-40% of rectal swab cultures from healthy infants, children, and adult women. Vertical transmission from mother to infant was the most common means of aquiring K1 organisms in term infants. Premature babies in a nursery with little maternal contact aquired K1 strains later then did term infants, and this aquisition may have been related to carriage by nursery staff. Capsular content and fermentation reactions of cerebrospinal-fluid K1 organisms were comparable to those found in rectal strains from healthy individuals. E. coli K1 with identical O and H antigens were found in maternal and infantile cultures of babies with E. coli meningitis. It seems very likely that host immune mechanisms play a significant role in the pathogenesis of neonatal E. coli K1 meningitis.
Records have been kept prospectively in our institution since 1928 of all positive blood cultures taken from neonates. Using a modification of objective Centers for Disease Control criteria to define sepsis, we reviewed the records of all neonates with positive blood cultures for the years 1979 to 1988 inclusive and found 270 cases of sepsis. The sepsis rate for infants less than or equal to 30 days of age was 2.7 cases/1000 live births, with a mortality rate from sepsis of 15.9%. There was an increase in sepsis due to commensal species (CS) over the period (P less than 0.007). The number of infants in the nursery who developed sepsis when more than 30 days of age also increased (P less than 0.002), as did the rate of sepsis from CS in this group (P less than 0.001). Isolation of CS from the blood with fulfillment of the modified Centers for Disease Control criteria was associated with a 13.7% mortality rate, whereas isolation of CS without fulfillment was associated with a 4% rate (P less than 0.01).
Mouse hybridoma antibodies (IgG and IgM) to O side chain determinants of Escherichia coli strain Bart (O18ac:K1:H7) were evaluated for their in vitro and in vivo activities against E. coli strains. Both IgG and IgM were opsonic in vitro and protected newborn rats challenged with a K1 E. coli strain, but their activities were strain specific. The antibodies protected against a K1 strain possessing a homologous O
serotype but not against one possessing a heterologous O serotype. These antibodies were not effective against the K5-encapsulated
O18 E. coli strain (possessing a homologous O type) but protected against its unencapsulated derivative. The opsonic and protective activities
of these antibodies were significantly greater with IgG than IgM. Both IgG and IgM, however, required complement for their
activities. When IgM to lipopolysaccharide was given to newborn rats in conjunction with IgM monoclonal antibody to the group
B meningococcal polysaccharide, the protective effect was significantly greater than that of either antibody alone. Combinations
of two (or more) antibodies to different cell wall components may be more beneficial in preventing and treating E. coli infection.
Sixty-three Escherichia coli strains isolated from neonatal sepsis or meningitis were studied and compared with previous data on fecal or urinary pyelonephritis-associated isolates from children. Characteristics significantly associated with neonatal infection were capsular type K1 (54%), O group 18 (27%), rough-type lipopolysaccharide together with K1 capsule (19%), and S fimbriae (29%). Within the neonatal infection group, the K1 capsule and rough lipopolysaccharide were most common among the youngest infants (0 to 21 days old) and in meningitis. Hemolysin production, P fimbriae, and X adhesions (adhesions not identifiable as type 1, P, or S) were significantly more common in the two materials from infections as compared with the fecal isolates. One large clone of 11 strains (O18:K1:H7, with both type 1 and S fimbriae) and three smaller ones (O7:K1:H1 and O6:K2:H1, both with type 1 and P fimbriae and X adhesions; and R:K1:H33 with no adhesions) were identified among the strains from neonatal infections. Only O6:K2:H1 strains were also common among the strains from pyelonephritis.
The structurally similar polysialic acid capsules of group B meningococci and Escherichia coli K1 are poor immunogens, and attempts are currently being made to improve their immunogenicity by chemical modifications. An IgG monoclonal antibody to these polysialic acid capsules was used for the study of the presence of structurally similar components in tissue glycoproteins to investigate the reasons for the poor immunogenicity and to evaluate potential dangers in active or passive immunization. By immunoblotting polysialic acid was detected outside the brain in newborn rat kidney, heart, and muscle. It appeared in immunoblots as one component and with similar mobility to the neural cell adhesion molecule N-CAM. Specificity studies of the antibody and endosialidase treatment showed that the polysialic acid glycans detected were composed of chains as long as eight sialic acid residues or more. The polysialic acid was not detected in the corresponding tissues of the adult animal. These results indicate that polysialic acid units are developmentally regulated components of both neural and extraneural tissues, and are bound to components with properties similar to a known cell-adhesion molecule. This together with the presence of low amounts of polysialic acid even in the adult brain, suggests potential hazards in vaccination trials and suggested immunotherapy of meningitis caused by group B meningococci or E. coli K1, which should be carefully assessed.
To define the relative roles of capsule and lipopolysaccharide in the virulence of Escherichia coli obtained from blood, we compared the behavior of K1- and K5-encapsulated strains in serum bactericidal and rat virulence
assays. Unencapsulated isogenic mutants selected from five parent strains of E. coli O12:K1, but not of O18:K1 or O7:K1 (all rough-specific phage insensitive), were lysed by normal human sera. In contrast,
isogenic mutants from strains of serotypes O6:K5 and O18:K5 retained the serum resistance of the parent strains. There was
a >105 difference in LD50 in newborn rats between K1-positive and K1-negative pairs of E. coli serotypes O18 and O7 and a >1 log difference between isogenic pairs of serotype 012; however, the K5 isogenic pairs had a
similar LD50. Some non-O6 O serotypes, however, required the K5 capsule for serum resistance. We conclude that some O serotypes require
encapsulation for optimal virulence but that other O serotypes may not.
In a search for more effective antimicrobial therapy of neonatal Escherichia coli infection, newer beta-lactam antibiotics,
cefotaxime and imipenem, were evaluated for their activities against a K1 E. coli strain in vitro and in vivo, and the results
were compared with those of conventional therapeutic regimens for neonatal E. coli infection: ampicillin-gentamicin and ampicillin-chloramphenicol.
Measured by MICs and MBCs, cefotaxime and imipenem were 8- to 512-fold more active in vitro than the older agents. For in
vivo studies, the following daily doses were used: 50 mg/kg for each of imipenem and cilastatin; 100 mg/kg for each of cefotaxime,
ampicillin, and chloramphenicol; and 10 mg/kg for gentamicin. At these doses, the mean bactericidal titers in blood and cerebrospinal
fluid were significantly greater with newer agents than with ampicillin-gentamicin and ampicillin-chloramphenicol. However,
at the doses used, the newer agents were not more effective in vivo than the older agents. This was shown by the similarities
in clearance of bacteria from blood and cerebrospinal fluid, incidences of meningitis in bacteremic animals, and mortality
rates. Thus, although these two newer antibiotics are more active in vitro and produce greater bactericidal titers in vivo,
they do not appear to be superior to conventional regimens for treatment of neonatal E. coli bacteremia and meningitis.
Examination of 77 strains of Escherichia coli from the cerebrospinal fluid of neonates with meningitis revealed 65 (84 per cent) with the capsular (K1) polysaccharide. The Esch. coli K1 capsular antigen has been shown to be immunochemically identical to the meningococcal Group B polysaccharide. These cerebrospinal-fluid Esch. coli K1 strains were associated with at least seven different somatic (O) and three flagella (H) antigens. In contrast, Esch. coli K1 strains were found in 14 of 36 (39 per cent) blood cultures of neonates without meningitis and in approximately 15 per cent of blood, urine and stool cultures from adults and rectal cultures from infants. The mean median lethal dose in a mucin-enhanced mouse model for 31 neonatal cerebrospinal-fluid K1 isolates was 168 organisms. In contrast, the median lethal dose for 10 neonatal non-K1 isolates was 58,000 and was greater than 10,000 organisms for six non-K1 enteropathogenic strains and five K1 strains isolated from normal infant stools. The ...
Bacterial counts in blood of E. coli (number of viable organisms per milliliter) are reported for 30 neonates whowere cultured because of suspected septicemia. Fifteen of the infants died; 13 of the deaths occurred within 48 hours after cultures were obtained and appropriate antibiotic therapy had been initiated. Thirty-five positive cultures were obtained from the 30 neonates; in 11 (31%) of the cultures the colony counts were in excess of 1,000/ml; in 5 (14%), the counts ranged from 50 to 1,000/ml; in 11 (31%), from 5 to 49/ml; in 8 (23%), from 0 to 4/ml. Seventy-three per cent (8/11) of the neonates with colony counts greater than 1,000/ml died.
The binding of pyelonephritogenic Escherichia coli strains to human uroepithelial cells from patients with and without P blood group antigens was investigated. Uroepithelial cells from p phenotypes bound pyelonephritogenic e. coli to a significantly lesser extent than did cells from P1 and P2 phenotypes. The binding of pyelonephritogenic E. coli to urinary epithelial cells of P1 phenotypes was blocked by the synthetic disaccharide alpha-D-Galp-(1 leads to 4)-beta-D-Galp whose structures is related to that of the P blood group antigens. Coating of P1 cells with a synthetic disaccharide derivative increased the binding of bacteria. None of 30 individuals of p phenotype had had urinary tract infection. The findings show that the disaccharide alpha-D-Galp-(1 leads to 4)-beta-D-Galp, previously shown to be the erythrocyte receptor for the fimbriae of pyelonephritongenic E. coli, is also the receptor structure on uroepithelial cells.
One hundred and thirty-one strains of Escherichia coli isolated from the cerebrospinal fluid of patients in the United Kingdom were tested for resistance to 13 antimicrobial drugs. Sixty-four strains (49%) were resistant to one or more drugs and 44 (34%) were resistant to three or more drugs. Resistance to ampicillin, sulphonamides, streptomycin and tetracycline occurred most frequently.
Since a limited number of 0 serogroups account for nearly 70% of bacteremic and meningitic Escherichia coli isolates, a polyvalent vaccine was made by conjugating a Pseudomonas aeruginosa exotoxin A carrier protein to the 0 polysaccharide of 12 serogroups of E. coli (01,02,04,06–08,012,015,016,018,025,075). No serious reactions occurred in 88 vaccinees. Fourfold or greater increases in
ELISA antibody levels over baseline were greatest (>60% of vaccinees) for 01,02,06–08 and 015; intermediate (∼50%) for 018
and 075, and poorest (≥45%) for 04, 012, 016, and 025. Responses with functionally active opsonophagocytic antibody generally
paralleled ELISA antibody responses. With the availability of a safe, immunogenic E. coli vaccine, active and passive immunization strategies merit further development as adjunctive treatment for E. coli bacteremia and neonatal meningitis.
Most cases of neonatal Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. In an attempt to identify E. coli structures contributing to invasion into the central nervous system (CNS), TnphoA mutagenesis was performed with an invasive CSF isolate of E. coli K1 strain RS218 (O18:K1:H7), and TnphoA mutants were examined for their noninvasive capability in brain microvascular endothelial cells (BMEC). The noninvasive mutants exhibited the invasive ability of < 1% of the parent strain. One of the noninvasive mutants (10A-23) with a single TnphoA insertion and no changes in phenotypic characteristics was found to be significantly less invasive into the CNS in the newborn rat model of hematogenous E. coli meningitis. The TnphoA inserts with flanking sequences were cloned and sequenced. A novel open reading frame (8.2 kDa) was identified. Open reading frame analysis indicated that the 8.2-kDa protein (Ibe10) contained multiple transmembrane domains. ibe10 was cloned into an expression vector, pQE30, and the purified Ibe10 was shown to inhibit invasion of BMEC by strain RS218. These findings indicate that ibe10 is one of the E. coli genes involved in the invasion of BMEC in vitro and in vivo.
To assess the role of S fimbriae in the pathogenesis of Escherichia coli meningitis, transformants of E. coli strains with or without S fimbriae plasmid were compared for their binding to microvessel endothelial cells isolated from bovine brain cortices (BMEC). The BMEC's displayed a cobblestone appearance, were positive for factor VIII, carbonic anhydrase IV, took up fluorescent-labeled acetylated low density lipoprotein, and exhibited gamma glutamyl transpeptidase activity. Binding of S fimbriated E. coli to BMEC was approximately threefold greater than nonfimbriated E. coli Similarly S fimbriated E. coli bound to human brain endothelial cells approximately threefold greater than nonfimbriated E. coli. Binding was reduced approximately 60% by isolated S fimbriae and about 80% by anti-S adhesin antibody. Mutating the S adhesin gene resulted in a complete loss of the binding, whereas mutagenesis of the major S fimbriae subunit gene sfaA did not significantly affect binding. Pretreatment of BMEC with neuraminidase or prior incubation of S fimbriated E. coli with NeuAc alpha 2,3-sialyl lactose completely abolished binding. These findings indicate that S fimbriated E. coli bind to NeuAc alpha 2,3-galactose containing glycoproteins on brain endothelial cells via a lectin-like activity of SfaS adhesin. This might be an important early step in the penetration of bacteria across the blood-brain barrier in the development of E. coli meningitis.
We reviewed our experience with gram-negative enteric bacillary meningitis in neonates and infants from 1969 through 1989. Ninety-eight patients were identified. Their ages were from 1 day to 2 years with a median of 10 days. In 25 patients (26%), predisposing factors were identified, the most common of which were neural tube defects and urinary tract anomalies. The causative agents were Escherichia coli (53%), Klebsiella-Enterobacter species (16%), Citrobacter diversus (9%), Salmonella species (9%), Proteus mirabilis (4%), Serratia marcescens (3%), Bacteroides fragilis (3%), and Aeromonas species (2%). At the time of diagnosis, Gram-stained smears of cerebrospinal fluid revealed gram-negative bacilli in 61% of patients. The causative organism was cultured from blood obtained from 55% of patients, and 21% had positive urine culture results. The cerebrospinal fluid leukocyte counts ranged from 0 to 80,600 cells/mm3, and the cerebrospinal fluid/serum glucose concentration ratio was less than 0.5 in 72% of patients. Antimicrobial regimens varied greatly. After initiation of antibiotic therapy, an average of 3 days was needed for eradication of bacteria from cerebrospinal fluid. The case-fatality rate was 17%, and 61% of survivors had long-term sequelae that included seizure disorders, hydrocephalus, physical disability, developmental delay, and hearing loss.
Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis. We have shown previously that outer membrane protein A (OmpA) contributes to E. coli K1 membrane invasion of brain microvascular endothelial cells. In this study we report that this OmpA+ K1 E. coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin. The specificity of the interaction between OmpA and GlcNAc beta 1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E. coli K1 invasion. Of interest, OmpA+ E. coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with alpha-fucosidase, suggesting that the GlcNAc beta 1-4GlcNAc moieties might be substituted with L-fucose on these cells. More importantly, the chitooligomers prevented entry of E. coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E. coli meningitis, suggesting that the GlcNAc beta 1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E. coli K1 across the blood-brain barrier. A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E. coli meningitis.