Interleukin-22 Contributes to Blood-Brain Barrier Disruption via STAT3/VEGFA Activation in Escherichia coli Meningitis.

Escherichia coli continues to be the predominant Gram-negative pathogen causing neonatal meningitis worldwide. Inflammatory mediators have been implicated in the pathogenesis of meningitis and are key therapeutic targets. The role of interleukin-22 (IL-22) in various diseases is diverse, with both protective and pathogenic effects. However, little is understood about the mechanisms underlying the damaging effects of IL-22 on the blood-brain barrier (BBB) in E. coli meningitis. We observed that meningitic E. coli infection induced IL-22 expression in the serum and brain of mice. The tight junction proteins (TJPs) components ZO-1, Occludin, and Claudin-5 were degraded in the mouse brain and human brain microvascular endothelial cells (hBMEC) following IL-22 administration. Moreover, the meningitic E. coli-caused increase in BBB permeability in wild-type mice was restored by knocking out IL-22. Mechanistically, IL-22 activated the STAT3-VEGFA signaling cascade in E. coli meningitis, thus eliciting the degradation of TJPs to induce BBB disruption. Our data indicated that IL-22 is an essential host accomplice during E. coli-caused BBB disruption and could be targeted for the therapy of bacterial meningitis.

CD48 and α7 Nicotinic Acetylcholine Receptor Synergistically Regulate FimH-Mediated Escherichia coli K1 Penetration and Neutrophil Transmigration Across Human Brain Microvascular Endothelial Cells.

FimH-mediated bacterial invasion and polymorphonuclear neutrophil (PMN) transmigration across human brain microvascular endothelial cells (HBMECs) are required for the pathogenesis of Escherichia coli meningitis. However, the underlying mechanism remains unclear. This study demonstrated that the TnphoA mutant (22A33) and FimH-knockout mutant (ΔFimH) of E coli strain E44, which resulted in inactivation of FimH, were less invasive and less effective in promoting PMN transmigration than their wild-type strain. FimH protein induced PMN transmigration, whereas calmodulin inhibitor significantly blocked this effect. Moreover, immunofluorescence and co-immunoprecipitation analysis indicated that colocalized CD48 and α7 nAChR formed a complex on the surface of HBMECs that is associated with increased cofilin dephosphorylation, which could be remarkably enhanced by FimH+ E44. Our study concluded that FimH-induced E coli K1 invasion and PMN migration across HBMECs may be mediated by the CD48-α7nAChR complex in lipid rafts of HBMEC via Ca2+ signaling and cofilin dephosphorylation.

New insights into meningitic Escherichia coli infection of brain microvascular endothelial cells from quantitative proteomics analysis.

BACKGROUND: Bacterial meningitis remains a big threat to the integrity of the central nervous system (CNS), despite the advancements in antimicrobial reagents. Escherichia coli is a bacterial pathogen that can disrupt the CNS function, especially in neonates. E. coli meningitis occurs after bacteria invade the brain microvascular endothelial cells (BMECs) that form a direct and essential barrier restricting the entry of circulating microbes and toxins to the brain. Previous studies have reported on several cellular proteins that function during meningitic E. coli infections; however, more comprehensive investigations to elucidate the potential targets involved in E. coli meningitis are essential to better understand this disease and discover new treatments for it. METHODS: The isobaric tags for relative and absolute quantification (iTRAQ) approach coupled with LC-MS/MS were applied to compare and characterize the different proteomic profiles of BMECs in response to meningitic or non-meningitic E. coli strains. KEGG and gene ontology annotations, ingenuity pathways analysis, and functional experiments were combined to identify the key host molecules involved in the meningitic E. coli-induced tight junction breakdown and neuroinflammatory responses. RESULTS: A total of 13 cellular proteins were found to be differentially expressed by meningitic E. coli strains PCN033 and RS218, including one that was also affected by HB101, a non-meningitic E. coli strain. Through bioinformatics analysis, we identified the macrophage migration inhibitory factor (MIF), granzyme A, NF-κB signaling, and mitogen-activated protein kinase (MAPK) pathways as being biologically involved in the meningitic E. coli-induced tight junction breakdown and neuroinflammation. Functionally, we showed that MIF facilitated meningitic E. coli-induced production of cytokines and chemokines and also helped to disrupt the blood-brain barrier by decreasing the expression of tight junction proteins like ZO-1, occludin. Moreover, we demonstrated the significant activation of NF-κB and MAPK signaling in BMECs in response to meningitic E. coli strains, which dominantly determined the generation of the proinflammatory cytokines including IL-6, IL-8, TNF-α, and IL-1ß. CONCLUSIONS: Our work identified 12 host cellular targets that are affected by meningitic E. coli strains and revealed MIF to be an important contributor to meningitic E. coli-induced cytokine production and tight junction disruption, and also the NF-κB and MAPK signaling pathways that are mainly involved in the infection-induced cytokines production. Characterization of these distinct proteins and pathways in BMECs will facilitate further elucidation of meningitis-causing mechanisms in humans and animals, thereby enabling the development of novel preventative and therapeutic strategies against infection with meningitic E. coli.

Escherichia coli K1 Modulates Peroxisome Proliferator-Activated Receptor γ and Glucose Transporter 1 at the Blood-Brain Barrier in Neonatal Meningitis.

Escherichia coli K1 meningitis continues to be a major threat to neonatal health. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with endothelial cell glycoprotein 96 (Ecgp96) in the blood-brain barrier to enter the central nervous system. Here we show that the interaction between OmpA and Ecgp96 downregulates peroxisome proliferator-activated receptor γ (PPAR-γ) and glucose transporter 1 (GLUT-1) levels in human brain microvascular endothelial cells, causing disruption of barrier integrity and inhibition of glucose uptake. The suppression of PPAR-γ and GLUT-1 by the bacteria in the brain microvessels of newborn mice causes extensive pathophysiology owing to interleukin 6 production. Pretreatment with partial or selective PPAR-γ agonists ameliorate the pathological outcomes of infection by suppressing interleukin 6 production in the brain. Thus, inhibition of PPAR-γ and GLUT-1 by E. coli K1 is a novel pathogenic mechanism in meningitis, and pharmacological upregulation of PPAR-γ and GLUT-1 levels may provide novel therapeutic avenues.

Serotype O18 avian pathogenic and neonatal meningitis Escherichia coli strains employ similar pathogenic strategies for the onset of meningitis.

Neonatal meningitis Escherichia coli K1 (NMEC) are thought to be transmitted from mothers to newborns during delivery or by nosocomial infections. However, the source of E. coli K1 causing these infections is not clear. Avian pathogenic E. coli (APEC) have the potential to cause infection in humans while human E. coli have potential to cause colibacillosis in poultry, suggesting that these strains may lack host specificity. APEC strains are capable of causing meningitis in newborn rats; however, it is unclear whether these bacteria use similar mechanisms to that of NMEC to establish disease. Using four representative APEC and NMEC strains that belong to serotype O18, we demonstrate that these strains survive in human serum similar to that of the prototypic NMEC strain E44, a derivative of RS218. These bacteria also bind and enter both macrophages and human cerebral microvascular endothelial cells (HCMEC/D3) with similar frequency as that of E44. The amino acid sequences of the outer membrane protein A (OmpA), an important virulence factor in the pathogenesis of meningitis, are identical within these representative APEC and NMEC strains. Further, these strains also require FcγRI-α chain (CD64) and Ecgp96 as receptors for OmpA in macrophages and HCMEC/D3, respectively, to bind and enter these cells. APEC and NMEC strains induce meningitis in newborn mice with varying degree of pathology in the brains as assessed by neutrophil recruitment and neuronal apoptosis. Together, these results suggest that serotype O18 APEC strains utilize similar pathogenic mechanisms as those of NMEC strains in causing meningitis.

Cytotoxic necrotizing factor 1 contributes to Escherichia coli meningitis.

E. coli is the most common Gram-negative bacteria causing neonatal meningitis, and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Recent reports of E. coli meningitis caused by antimicrobial resistant strains are a particular concern. These findings indicate that a novel strategy is needed to identify new targets for prevention and therapy of E. coli meningitis. Cytotoxic necrotizing factor 1 (CNF1) is a bacterial virulence factor associated principally with E. coli strains causing urinary tract infection and meningitis. We have shown that CNF1 contributes to E. coli invasion of the blood-brain barrier and penetration into the brain, the essential step in the development of E. coli meningitis, and identified the host receptor for CNF1, 37-kDa laminin receptor precursor (37LRP). CNF1, however, is a cytoplasmic protein and its contribution to E. coli invasion of the blood-brain barrier requires its secretion from the bacterial cytoplasm. No signal peptide is found in the CNF1 sequence. CNF1 secretion is, therefore, a strategy utilized by meningitis-causing E. coli to invade the blood-brain barrier. Elucidation of the mechanisms involved in CNF1 secretion, as shown in this report with the involvement of Fdx and YgfZ provides the novel information on potential targets for prevention and therapy of E. coli meningitis by virtue of targeting the secretion of CNF1.

A conserved virulence plasmidic region contributes to the virulence of the multiresistant Escherichia coli meningitis strain S286 belonging to phylogenetic group C.

Recent isolation of the non-K1 Escherichia coli neonatal meningitis strain S286, belonging to phylogroup C, which is closely related to major group B1, and producing an extended-spectrum beta-lactamase, encouraged us to seek the genetic determinants responsible for its virulence. We show that S286 belongs to the sequence O type ST23O78 and harbors 4 large plasmids. The largest one, pS286colV (~120 kb), not related to resistance, contains genes characteristic of a Conserved Virulence Plasmidic (CVP) region initially identified in B2 extra-intestinal avian pathogenic E. coli (APEC) strains and in the B2 neonatal meningitis E. coli strain S88. The sequence of this CVP region has a strong homology (98%) with that of the recently sequenced plasmid pChi7122-1 of the O78 APEC strain Chi7122. A CVP plasmid-cured variant of S286 was less virulent than the wild type strain in a neonatal rat sepsis model with a significant lower level of bacteremia at 24 h (4.1 ± 1.41 versus 2.60 ± 0.16 log CFU/ml, p = 0.001) and mortality. However, the mortality in the model of adult mice was comparable between wild type and variant indicating that pS286colV is not sufficient by itself to fully explain the virulence of S286. Gene expression analysis of pS286colV in iron depleted environment was very close to that of pS88, suggesting that genes of CVP region may be expressed similarly in two very different genetic backgrounds (group C versus group B2). Screening a collection of 178 human A/B1 extraintestinal pathogenic E. coli (ExPEC) strains revealed that the CVP region is highly prevalent (23%) and MLST analysis indicated that these CVP positive strains belong to several clusters and mostly to phylogroup C. The virulence of S286 is explained in part by the presence of CVP region and this region has spread in different clusters of human A/B1 ExPEC, especially in group C.

Neuropathogenic Escherichia coli K1 does not exhibit proteolytic activities to exert its pathogenicity.

BACKGROUND: Proteases are well-known virulence factors that promote survival, pathogenesis and immune evasion of many pathogens. Several lines of evidence suggest that the blood-brain barrier permeability is a prerequisite in microbial invasion of the central nervous system. Because proteases are frequently associated with vascular permeability by targeting junctional proteins, here it is hypothesized that neuropathogenic Escherichia coli K1 exhibit proteolytic activities to exert its pathogenicity. METHODS: Zymographic assays were performed using collagen and gelatin as substrates. The lysates of whole E. coli K1 strain E44, or E. coli K-12 strain HB101 were tested for proteolytic activities. The conditioned media were prepared by incubating bacteria in RPMI-1640 in the presence or absence of serum. The cell-free supernatants were collected and tested for proteases in zymography as mentioned above. Additionally, proteolytic degradation of host immune factors was determined by co-incubating conditioned media with albumin/immunoglobulins using protease assays. RESULTS: When collagen or gelatin were used as substrates in zymographic assays, neither whole bacteria nor conditioned media exhibited proteolytic activities. The conditioned media of neuropathogenic E. coli K1 strain E44, or E. coli K-12 strain HB101 did not affect degradation of albumin and immunoglobulins using protease assays. CONCLUSIONS: Neither zymographic assays nor protease assays detected proteolytic activities in either the whole bacteria or conditioned media of E. coli K1 strain E44 and E. coli K-12 strain HB101. These findings suggest that host cell monolayer disruptions and immune evasion strategies are likely independent of proteolytic activities of neuropathogenic E. coli K1.

[Clinical analysis of 31 cases of neonatal purulent meningitis caused by Escherichia coli].

OBJECTIVE: Neonatal purulent meningitis is a severe infection responsible for high mortality and disabling sequelae. Escherichia coli is the main pathogen of neonatal purulent meningitis. This study explored the clinical characteristics and antibiotic resistance of Escherichia coli-induced neonatal meningitis. METHODS: A retrospective chart review was performed. A total of 31 cases of neonatal purulent meningitis caused by Escherichia coli were identified in the neonatal intensive care unit between January 1, 2001 and December 31, 2011. The clinical characteristics and antibiotic sensitivity test results were analyzed. RESULTS: Fever, poor feeding, lethargy and seizure were common clinical signs of neonatal purulent meningitis caused by Escherichia coli. Acute complications mainly included hyponatremia (17 cases), hydrocephalus (8 cases), subdural collection (2 cases), ventriculitis (2 cases) and cerebral infarction (1 case). Thirty neonates (97%) had increased CRP levels. Of the 31 patients, 14 cases were cured and 12 had adverse outcomes (5 patients died during hospitalization). Escherichia coli strains were resistant (>50%) to commonly used penicillins and cephalosporins between 2007 and 2011, presenting significantly higher resistance rates than between 2001 and 2006. The detection rate of extended spectrum ß-lactamases (ESBLs)-producing strains between 2007 and 2011 increased significantly compared with between 2001 and 2006 (57% vs 0). CONCLUSIONS: The clinical manifestations of neonatal purulent meningitis caused by Escherichia coli are non specific. The outcome is poor. Monitoring of CRP levels is valuable for the early diagnosis of neonatal purulent meningitis. The antimicrobial resistance rates of Escherichia coli are increasing, especially to cephalosporins. The percentage of ESBLs-producing strains is increasing over the years.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Suppurative meningitis in a 7-day-old Formosan sambar deer (Cervus unicolor swinhoei) caused by Escherichia coli.

This article describes the clinical and pathological features of an orphan 7-day-old, male Formosan sambar fawn that was hospitalized for treatment of weakness. The fawn had been deprived of colostrum and developed suppurative meningitis that was attributed to Escherichia coli.

[A serious and unexpected infectious complication after transrectal prostate biopsy]. / Une complication infectieuse grave inattendue après biopsie transrectale de prostate.

Serious infectious complications may appear after a surgical procedure despite antimicrobial prophylaxis. We report a case of Escherichia Coli meningitis after a prostate puncture biopsy. Many factors may have contributed to this rare complication such as fluoroquinolone resistance, and/or the bacterium's ability to cross the brain blood barrier. The patient was not found susceptible to severe infections.

No neuroprotective effect of erythropoietin under clinical treatment conditions in a rabbit model of Escherichia coli meningitis.

Despite effective antibiotic treatment, neuronal injury is frequent among children and adults with bacterial meningitis resulting in a high rate of death and neurologic sequelae. The hematopoietic cytokine erythropoietin (EPO) provides neuroprotection in models of acute and chronic neurologic diseases. We studied whether recombinant EPO (rEPO) reduces neuronal damage in a rabbit model of Escherichia coli meningitis. Inflammation within the central nervous system (CNS) was monitored by measurement of bacterial load, pleocytosis, protein, and lactate in the cerebrospinal fluid (CSF). Neuronal damage was measured by quantification of the density of apoptotic neurons in the hippocampal dentate gyrus and the concentration of the global neuronal destruction marker neuron-specific enolase (NSE) in CSF. To increase clinical relevance, rEPO was applied as adjunctive therapy from the beginning of antibiotic therapy 12 h after infection. EPO treatment applied as an intravenous injection at a dose of 1000 IU/kg body weight resulted in plasma concentrations of 6993 +/- 1406 mIU/mL, CSF concentrations of 1291 +/- 568 mIU/mL, and a CSF-to-plasma ratio of 0.18 +/- 0.07 (mean +/- SD) 6 h after injection. Under these treatment conditions, no anti-inflammatory or neuroprotective effect of EPO was observed. "

Cranial MRI of small rodents using a clinical MR scanner.

Increasing numbers of small animal models are in use in the field of neuroscience research. Magnetic resonance imaging (MRI) provides an excellent method for non-invasive imaging of the brain. Using three-dimensional (3D) MR sequences allows lesion volumetry, e.g. for the quantification of tumor size. Specialized small-bore animal MRI scanners are available for high-resolution MRI of small rodents' brain, but major drawbacks of this dedicated equipment are its high costs and thus its limited availability. Therefore, more and more research groups use clinical MR scanners for imaging small animal models. But to achieve a reasonable spatial resolution at an acceptable signal-to-noise ratio with these scanners, some requirements concerning sequence parameters have to be matched. Thus, the aim of this paper was to present in detail a method how to perform MRI of small rodents brain using a standard clinical 1.5 T scanner and clinically available radio frequency coils to keep material costs low and to circumvent the development of custom-made coils.

Severe progressive late onset myelopathy and arachnoiditis following neonatal meningitis.

This case series describes four children who had meningitis in the neonatal period. After a stable period of years, they developed a myelopathy caused by chronic arachnoiditis. The myelopathy was precipitated by a fall in two cases, and in two cases there was an acute deterioration after surgery. A history of neonatal meningitis should be taken into consideration before planning surgery or anaesthesia. Careful intra-operative positioning, immobilisation of the neck, and maintenance of blood pressure is important but may not prevent this complication.

Effects of hypertonic (7%) saline on brain injury in experimental Escherichia coli meningitis.

We sought to know whether hypertonic (7%) saline (HTS) attenuates brain injury by improving cerebral perfusion pressure (CPP) and down-modulating acute inflammatory responses in experimental bacterial meningitis in the newborn piglet. Twenty-five newborn piglets were assorted into three groups: 6 in the control group (C), 10 in the meningitis group (M), and 9 in the meningitis with HTS infusion group (H). Meningitis was induced by intracisternal injection of 10(8) colony forming units of Escherichia coli in 100 microL of saline. 10 mL/kg of HTS was given intravenously as a bolus 6 hr after induction of meningitis, thereafter the infusion rate was adjusted to maintain the serum sodium level between 150 and 160 mEq/L. HTS significantly attenuated meningitis-induced brain cell membrane disintegration and dysfunction, as indicated by increased lipid peroxidation products and decreased Na+, K+-ATPase activity in the cerebral cortex in M. HTS significantly attenuated acute inflammatory markers such as increased intracranial pressure, elevated lactate level and pleocytosis in the cerebrospinal fluid observed in M. Reduced CPP observed in M was also significantly improved with HTS infusion. These findings implicate some attenuation of the meningitis-induced alterations in cerebral cortical cell membrane structure and function with HTS, possibly by improving CPP and attenuating acute inflammatory responses.

Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-kappaB activation.

Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-alpha, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1beta, IL-1beta, and IL-8). In contrast, infection of monocytes with a mutant E. coli, which lacks outer membrane protein A (OmpA- E. coli) resulted in robust production of cytokines and chemokines. Wild-type E. coli K1 (OmpA+ E. coli) prevented the phosphorylation and its degradation of inhibitor of kappaB, thereby blocking the translocation of nuclear factor (NF)-kappaB to the nucleus. OmpA+ E. coli-infected cells, subsequently subjected to lipopolysaccharide challenge, were crippled severely in their ability to activate NF-kappaB to induce cytokine/chemokine production. Selective inhibitors of the extracellular signal-regulated kinase (ERK) 1/2 pathway and p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase, significantly reduced the activation of NF-kappaB and the production of cytokines and chemokines induced by OmpA- E. coli, indicating a role for these kinases in the NF-kappaB/cytokine pathway. It is interesting that the phosphorylation of ERK 1/2 and p38 MAPK was notably reduced in monocytes infected with OmpA+ E. coli when compared with monocytes infected with OmpA- E. coli, suggesting that the modulation of upstream events common for NF-kappaB and MAPKs by the bacterium is possible. The ability of OmpA+ E. coli K1 to inhibit the macrophage response temporarily may enable bacterial survival and growth within the host for the onset of meningitis by E. coli K1.

[Pulmonary strongyloidiasis complicated by E. coli meningitis in a HIV-1 and HTLV-1 positive patient]. / Anguillulose pulmonaire compliquée d'une méningite à E. coli chez un patient VIH1 et HTLV1 positif.

INTRODUCTION: Disseminated strongyloidiasis occurs in immunodepressed patients, notably those infected by retroviruses. OBSERVATION: A pulmonary strongyloidiasis, complicated by an Escherichia coli meningitis, occurred in a patient exhibiting seropositivity HIV1 for the past year. The status of cell immunity, with 354 lymphocytes T CD4+/mm3, could not explain this severe complication. This led to the diagnosis of an HTLV1 infection. The strongyloidiasis was treated with two cycles of ivermectine, which cured the patient. COMMENTS: In HIV-infected patients exhibiting severe strongyloidiasis, research for an HTLV co-infection is recommended.

3-Aminobenzamide, a poly (ADP-ribose) synthetase inhibitor, attenuates the acute inflammatory responses and brain injury in experimental Escherichia coli meningitis in the newborn piglet.

The aim of the present study was to evaluate the anti-inflammatory and neuroprotective effects of a poly (ADP-ribose) synthetase inhibitor 3-aminobenzamide during the early phase of experimental bacterial meningitis in the newborn piglet. Meningitis was induced by intracisternal injection of 10(8) colony forming units of Escherichia coli in 100 microl of saline. 3-Aminobenzamide, given 30 mg kg(-1) as a bolus i.v. injection 30 min before induction of meningitis, significantly attenuated the meningitis-induced acute inflammatory responses such as increased cerebrospinal fluid (CSF) lactate concentration, CSF leukocytosis and increased CSF tumor necrosis factor-alpha level. However, meningitis-induced increase in intracranial pressure and decrease in CSF glucose level were not significantly improved. Increased cerebral cortical cell membrane lipid peroxidation products (conjugated dienes) and decreased brain ATP/phosphocreatine levels observed in the meningitis group were also significantly improved with 3-aminobenzamide treatment. However, the improvement of reduced Na+, K+-ATPase activity did not reach a statistical significance (p = 0.06). In summary, 3-aminobenzamide significantly attenuated the acute inflammatory responses and the ensuing brain injury during the early phase of neonatal bacterial meningitis. These findings suggest that poly (ADP-ribose) synthetase inhibitors such as 3-aminobenzamide might be a promising novel anti-inflammatory and neuroprotective adjuvant therapy in neonatal bacterial meningitis.