Taxonomic position, antibiotic resistance and virulence factor production by Stenotrophomonas isolates from patients with cystic fibrosis and other chronic respiratory infections.

BACKGROUND: The potential pathogenic role of Stenotrophomonas maltophilia in lung disease and in particular in cystic fibrosis is unclear. To develop further understanding of the biology of this taxa, the taxonomic position, antibiotic resistance and virulence factors of S. maltophilia isolates from patients with chronic lung disease were studied. RESULTS: A total of 111 isolates recovered between 2003 and 2016 from respiratory samples from patients in five different countries were included. Based on a cut-off of 95%, analysis of average nucleotide identity by BLAST (ANIb) showed that the 111 isolates identified as S. maltophilia by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) belonged to S. maltophilia (n = 65), S. pavanii (n = 6) and 13 putative novel species (n = 40), which each included 1-5 isolates; these groupings coincided with the results of the 16S rDNA analysis, and the L1 and L2 ß-lactamase Neighbor-Joining phylogeny. Chromosomally encoded aminoglycoside resistance was identified in all S. maltophilia and S. pavani isolates, while acquired antibiotic resistance genes were present in only a few isolates. Nevertheless, phenotypic resistance levels against commonly used antibiotics, determined by standard broth microbroth dilution, were high. Although putative virulence genes were present in all isolates, the percentage of positive isolates varied. The Xps II secretion system responsible for the secretion of the StmPr1-3 proteases was mainly limited to isolates identified as S. maltophilia based on ANIb, but no correlation with phenotypic expression of protease activity was found. The RPF two-component quorum sensing system involved in virulence and antibiotic resistance expression has two main variants with one variant lacking 190 amino acids in the sensing region. CONCLUSIONS: The putative novel Stenotrophomonas species recovered from patient samples and identified by MALDI-TOF/MS as S. maltophilia, differed from S. maltophilia in resistance and virulence genes, and therefore possibly in pathogenicity. Revision of the Stenotrophomonas taxonomy is needed in order to reliably identify strains within the genus and elucidate the role of the different species in disease.

Dynamics of Intestinal Carriage of Extended-Spectrum Beta-lactamase-Producing Enterobacteriaceae in the Dutch General Population, 2014-2016.

BACKGROUND: In the Netherlands, the prevalence of intestinal extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in community-dwelling subjects is ~5%. Little is known about the dynamics of ESBL-E carriage. METHODS: In a nationwide, population-based study (2014-2016) with 4177 community-dwelling subjects, fecal samples from 656 subjects were collected after 1 (time point [T] = 1) and 6 (T = 2) months. The growth of ESBL-E was quantified and a whole-genome sequence analysis was performed. Subjects were categorized as either an incidental, short-term, or long-term carrier or as a noncarrier. Risk factors were determined by random forest models and logistic regression. The transmissibility and duration of ESBL-E carriage was quantified using a transmission model, which also incorporated previous study data. RESULTS: Out of 656 participants, 96 were ESBL-E carriers at T = 0. Of these, 66 (10.1%) subjects were incidental carriers, 22 (3.3%) were short-term carriers, and 38 (5.8%) were long-term carriers; the remaining 530 (80.8%) were noncarriers. The risk factors for long-term carriage were travelling to Asia, swimming in a sea/ocean, and not changing the kitchen towel daily. The log-transformed colony forming units ratio at T = 0 was predictive for ESBL-E carriage at T = 1 (odds ratio [OR], 1.3; 95% confidence interval [CI], 1.2-1.6) and T = 2 (OR, 1.2; 95% CI, 1.1-1.4). Model simulations revealed a median decolonization rate of 2.83/year, an average duration of carriage of 0.35 years, and an acquisition rate of 0.34/year. The trend of the acquisition rate during the study period was close to 0. CONCLUSIONS: The risk factors for long-term ESBL-E carriage were travel- and hygiene-related. The dynamics of ESBL-E carriage in the general Dutch population are characterized by balancing decolonization and acquisition rates.

Prevalence, risk factors and genetic characterisation of extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae (ESBL-E and CPE): a community-based cross-sectional study, the Netherlands, 2014 to 2016.

BackgroundThe epidemiology of carriage of extended-spectrum beta-lactamase-producing (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) in the general population is unknown.AimIn this observational study, the prevalence and risk factors for intestinal ESBL-E and CPE carriage in the Dutch general population were determined. ESBL-E were characterised.MethodsFrom 2014 to 2016, ca 2,000 residents were invited monthly to complete a questionnaire and provide a faecal sample, which was tested for ESBL-E. The first 1,758 samples were also tested for CPE. Risk factors for ESBL-E carriage were identified by multivariable logistic regression analysis. ESBL-E isolates underwent whole genome sequencing.ResultsOf 47,957 individuals invited, 4,177 (8.7%) completed the questionnaire and provided a faecal sample. ESBL-E were detected in 186 (4.5%) individuals, resulting in an adjusted prevalence of 5.0% (95% confidence interval (CI):3.4-6.6%). Risk factors were: born outside the Netherlands (odds ratio (OR): 1.99; 95% CI: 1.16-4.54), eating in restaurants > 20 times/year (OR: 1.70; 95% CI: 1.04-2.76), antibiotic use < 6 months ago (OR: 2.05; 95% CI: 1.05-4.03), swimming in sea/ocean < 12 months ago (OR: 1.63; 95% CI: 1.11-2.39), travelling to Africa (OR: 3.03; 95% CI: 1.23-7.46) or Asia (OR: 2.00; 95% CI: 1.02-3.90) < 12 months ago, and not changing kitchen towels daily (OR: 2.19; 95% CI: 1.24-3.87). The last had the largest population attributable risk (PAR) (47.5%). Eighty-four of 189 (44.4%) ESBL-E isolates carried bla CTX-M-15. Escherichia coli isolates belonged to 70 different sequence types (ST)s, of which ST131 (42/178 isolates; 23.6%) was most prevalent. Associations were observed between IncFIA plasmids and ST131 and bla CTX-M-27, and between IncI1 and ST88 and bla CTX-M-1. No CPE were detected.ConclusionsThe prevalence of ESBL-E carriage in the Netherlands' community-dwelling population is 5.0%. Identified risk factors were mostly travelling (particularly to Asia and Africa) and kitchen hygiene. CPE were not detected.

Sequence-based epidemiology of an OXA-48 plasmid during a hospital outbreak.

Objectives. A large OXA-48 outbreak in the Netherlands involved the spread of OXA-48producing Enterobacteriaceae among at least 118 patients, suggesting horizontal transfer of this resistance gene through one or more plasmids. Elucidating transmission dynamics of resistance plasmids is hampered by the low resolution of classic typing methods. This study aimed to investigate the molecular epidemiology of plasmids carrying OXA-48 carbapenemase using a next-generation sequencing approach.Methods. A total of 68 OXA-48-producing Enterobacteriaceae isolated from the hospital outbreak, as well as 22 non-outbreak related OXA-48-producing Enterobacteriaceae from the Netherlands, Libya and Turkey were selected. Plasmids were sequenced using the Illumina Miseq platform, and read sets were assembled and analysed.Results. In all plasmids bla OXA-48 was embedded in transposon Tn1999.2 and located on a ca. 62 kb IncL/M conjugative plasmid in 14 different species. There were a maximum of 2 SNPs (single nucleotide polymorphisms) between the core sequence alignment of all plasmids. Closely related sequence variants of this plasmid were detected in non-outbreak isolates from the Netherlands and other countries. Thirty-one of 89 OXA-48-producing isolates also harboured bla CTX-M-15, which was not located on the bla OXA-48-carrying plasmid. Sequencing of four plasmids harbouring bla CTX-M15 revealed extensive plasmid heterogeneity.Conclusions. A ca 62 kb plasmid was responsible for the OXA-48 outbreak in a Dutch hospital. Our findings provide strong evidence for both within-host inter-species and between host dissemination of plasmid-based OXA-48 during a nosocomial outbreak. These findings exemplify the complex epidemiology of carbapenemase producing Enterobacteriaceae (CPE).

Ceftibuten plus amoxicillin-clavulanic acid for oral treatment of urinary tract infections with ESBL producing E. coli and K. pneumoniae: a retrospective observational case-series.

This study aimed to evaluate the clinical and bacteriological effect of oral treatment with ceftibuten plus amoxicillin-clavulanic acid in patients with a urinary tract infection (UTI) caused by an extended-spectrum ß-lactamase (ESBL)-producing micro-organism. In this retrospective observational case-series, oral treatment with ceftibuten 400 mg QD plus amoxicillin-clavulanic acid 625 mg TID for 14 days was evaluated in ten patients with pyelonephritis caused by an ESBL-positive micro-organism resistant to ciprofloxacin and co-trimoxazole. Presence of ESBL genes was confirmed using PCR and micro-array. EUCAST breakpoints were used for susceptibility testing. Ten patients (five women) were evaluated in 2016 and 2017. Six patients were from outpatient hospital care, and four from primary care. Urinary cultures yielded seven E. coli and three K. pneumoniae ESBL-positive isolates. Using Vitek-2, all isolates were resistant to cefotaxime, and resistant (n = 7) or intermediately susceptible (n = 3) to ceftazidime. With disc diffusion, all isolates were susceptible to ceftibuten (zones 25-32 mm), while with MIC test strips eight of ten isolates were resistant to ceftibuten (MICs 0.5-4 mg/L). An amoxicillin-clavulanic acid disc next to the ceftibuten disc extended the ceftibuten zone by 2-8 mm. All patients experienced clinical cure. Bacteriological cure (absence of pretreatment micro-organism in the first follow-up culture obtained within 3 months after treatment) was observed in all eight patients with follow-up cultures. This case-series shows that the synergistic combination of ceftibuten plus amoxicillin-clavulanic acid may be an option for oral treatment of UTIs caused by ESBL producing E. coli or K. pneumoniae.

Rapid and accurate detection of carbapenemase genes in Enterobacteriaceae with the Cepheid Xpert Carba-R assay.

Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae is pivotal for adequate antibiotic therapy and infection control. The Cepheid Xpert Carba-R assay detects and identifies the most prevalent carbapenemases (KPC, VIM, IMP, NDM and OXA-48), using automated real-time PCR. The test performance of the Xpert Carba-R was evaluated with 129 well-characterized non-repeat Enterobacteriaceae isolates, suspected for carbapenemase production, i.e. with meropenem MICs >0.25 mg l-1. The isolate collection contained 100 carbapenemase-producing isolates (36 KPC-2 or KPC-3, 20 VIM-1, 4 KPC-2 plus VIM-1, 5 NDM-1, 2 IMP-1, 1 IMP-28, 1 IMP-1 plus VIM-1 and 31 OXA-48 like) and 29 negative control isolates producing extended-spectrum ß-lactamase and/or AmpC ß-lactamases. PCR and sequencing of ß-lactamase genes were used as reference tests. The sensitivity of the Xpert Carba-R was 100 % (100/100), with a 100 % (29/29) specificity. The time to result was approximately 55 min with a hands-on time of only 1 min per isolate. In conclusion, the Carba-R assay is a rapid and accurate instrument for the confirmation and identification of the blaKPC, blaVIM, blaIMP, blaNDM and blaOXA-48 genes.

Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages.

Third-generation cephalosporins are a class of ß-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typing (PBRT). E coli strains were compared using Multi-Locus-Sequence-Typing (MLST). In 12 of 98 chicken meat samples (12%), pAmpC producing E. coli were detected (all blaCMY-2). Of the 479 human E. coli, 25 (5.2%) harboured pAmpC genes (blaCMY-2 n = 22, blaACT n = 2, blaMIR n = 1). PBRT showed that 91% of poultry meat isolates harboured blaCMY-2 on an IncK plasmid, and 9% on an IncI1 plasmid. Of the human blaCMY-2 producing isolates, 42% also harboured blaCMY-2 on an IncK plasmid, and 47% on an IncI1 plasmid. Thus, 68% of human pAmpC producing E. coli have the same AmpC gene (blaCMY-2) and plasmid type (IncI1 or IncK) as found in poultry meat. MLST showed one cluster containing one human isolate and three meat isolates, with an IncK plasmid. These findings imply that a foodborne transmission route of blaCMY-2 harbouring plasmids cannot be excluded and that further evaluation is required.

Detection of carbapenemase-producing Enterobacteriaceae with a commercial DNA microarray.

The Check-MDR CT102 DNA microarray enables detection of the most prevalent carbapenemases (NDM, VIM, KPC, OXA-48 and IMP) and extended-spectrum ß-lactamase (ESBL) gene families (SHV, TEM and CTX-M). The test performance of this microarray was evaluated with 95 Enterobacteriaceae isolates suspected of being carbapenemase producers, i.e. with meropenem MICs ≥0.5 mg l(-1). The collection of isolates contained 70 carbapenemase-producing isolates, including 37 bla(KPC)-, 20 bla(VIM)-, five bla(OXA-48)-, four bla(KPC)/bla(VIM)- and four bla(NDM)-positive isolates; and 25 carbapenemase-gene-negative isolates. ESBLs were produced by 51 of the isolates. PCR and sequencing of ß-lactamase genes was used as reference test. For detection of carbapenemases, the sensitivity of the microarray was 97% (68/70), with 100% specificity. The two negative isolates tested positive when the microarray test was repeated; these isolates were an OXA-48- and a KPC-producing isolate. For ESBL detection, the sensitivity was 100% (51/51) and the specificity was 98% (43/44), although 20% of the SHV-12 ESBLs were categorized as SHV-2-like ESBLs. In conclusion, the CDT102 microarray is a rapid and accurate tool for the detection of carbapenemase and ESBL genes, although the array seems less suitable for epidemiology of ESBL genes.

Comparison of ESBL contamination in organic and conventional retail chicken meat.

Contamination of retail chicken meat by Extended Spectrum Beta-Lactamase (ESBL) producing bacteria likely contributes to the increasing incidence of infections with these bacteria in humans. This study aimed to compare the prevalence and load of ESBL positive isolates between organic and conventional retail chicken meat samples, and to compare the distribution of ESBL genes, strain genotypes and co-resistance. In 2010, 98 raw chicken breasts (n=60 conventional; n=38 organic) were collected from 12 local stores in the Netherlands. Prevalence of ESBL producing micro-organisms was 100% on conventional and 84% on organic samples (p<0.001). Median loads of ESBL producing micro-organisms were 80 (range <20-1360) in conventional, and <20 (range 0-260) CFU/25 g in organic samples (p=0.001). The distribution of ESBL genes in conventional samples and organic samples was 42% versus 56%, respectively (N.S.), for CTX-M-1, 20% versus 42% (N.S.) for TEM-52, and 23% versus 3% (p<0.001) for SHV-12. CTX-M-2 (7%), SHV-2 (5%) and TEM-20 (3%) were exclusively found in conventional samples. Co-resistance rates of ESBL positive isolates were not different between conventional and organic samples (co-trimoxazole 56%, ciprofloxacin 14%, and tobramycin 2%), except for tetracycline, 73% and 46%, respectively, p<0.001). Six of 14 conventional meat samples harbored 4 MLST types also reported in humans and 5 of 10 organic samples harbored 3 MLST types also reported in humans (2 ST10, 2 ST23, ST354). In conclusion, the majority of organic chicken meat samples were also contaminated with ESBL producing E. coli, and the ESBL genes and strain types were largely the same as in conventional meat samples.

Population distribution of Beta-lactamase conferring resistance to third-generation cephalosporins in human clinical Enterobacteriaceae in the Netherlands.

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne ß-lactamases that confer resistance to third-generation cephalosporins. The epidemiology of these bacteria is not well understood, and was, therefore, investigated in a selection of 636 clinical Enterobacteriaceae with a minimal inhibitory concentration >1 mg/L for ceftazidime/ceftriaxone from a national survey (75% E. coli, 11% E. cloacae, 11% K. pneumoniae, 2% K. oxytoca, 2% P. mirabilis). Isolates were investigated for extended-spectrum ß-lactamases (ESBLs) and ampC genes using microarray, PCR, gene sequencing and molecular straintyping (Diversilab and multi-locus sequence typing (MLST)). ESBL genes were demonstrated in 512 isolates (81%); of which 446 (87%) belonged to the CTX-M family. Among 314 randomly selected and sequenced isolates, bla(CTX-M-15) was most prevalent (n = 124, 39%), followed by bla(CTX-M-1) (n = 47, 15%), bla(CTX-M-14) (n = 15, 5%), bla(SHV-12) (n = 24, 8%) and bla(TEM-52) (n = 13, 4%). Among 181 isolates with MIC ≥16 mg/L for cefoxitin plasmid encoded AmpCs were detected in 32 and 27 were of the CMY-2 group. Among 102 E. coli isolates with MIC ≥16 mg/L for cefoxitin ampC promoter mutations were identified in 29 (28%). Based on Diversilab genotyping of 608 isolates (similarity cut-off >98%) discriminatory indices of bacteria with ESBL and/or ampC genes were 0.994, 0.985 and 0.994 for E. coli, K. pneumoniae and E. cloacae, respectively. Based on similarity cut-off >95% two large clusters of E. coli were apparent (of 43 and 30 isolates) and 21 of 21 that were typed by belonged to ST131 of which 13 contained bla(CTX-M-15). Our findings demonstrate that bla(CTX-M-15) is the most prevalent ESBL and we report a larger than previously reported prevalence of ampC genes among Enterobacteriaceae responsible for resistance to third-generation cephalosporins.

A set of multiplex PCRs for genotypic detection of extended-spectrum ß-lactamases, carbapenemases, plasmid-mediated AmpC ß-lactamases and OXA ß-lactamases.

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to ß-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum ß-lactamases (ESBLs) are most widely disseminated, other ß-lactamase families have also recently emerged, such as plasmid-mediated AmpC ß-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent ß-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC ß-lactamases and OXA ß-lactamases.

Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells.

BACKGROUND: Mannose binding lectin (MBL) is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro. RESULTS: High avidity binding was observed between MBL and C. albicans and C. parapsilosis. Addition of MBL to MBL deficient serum increased the deposition of C4 and C3b and enhanced the uptake of C. albicans, C. parapsilosis and acapsular C. neoformans by polymorphonuclear cells (PMNs). Compared to other microorganisms, such as Escherichia coli, Staphylococcus aureus and Cryptococcus neoformans, C. parapsilosis and Candida albicans were potent activators of the lectin pathway. CONCLUSION: Our results suggest that MBL plays a crucial role in the innate immunity against infections caused by yeast by increasing uptake by PMN.

The Skn7 response regulator of Cryptococcus neoformans is involved in oxidative stress signalling and augments intracellular survival in endothelium.

Cryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis. There is accumulating evidence that C. neoformans is a facultative intracellular pathogen, residing in macrophages and endothelium. The molecular mechanism conferring resistance to phagolysosomal killing in these cells is a key unresolved issue. To gain insight into the fungal adaptive strategies, serial analysis of gene expression was used to map genes differentially expressed in an intraphagocytic environment. By comparing transcript profiles of C. neoformans serotype D B3501 cells recovered from endothelial cells with those from free-grown cryptococci, we identified the cryptococcal homologue of the SKN7 two-component stress response regulator gene from Saccharomyces cerevisiae. Studies with C. neoformans cells disrupted for SKN7 revealed an increased susceptibility to t-butyl hydroperoxide (100% lethality at 0.7 mM, vs. 1.0 mM for wild type) and significantly lower survival rates in endothelial infection experiments. Mice experiments revealed that SKN7 disruption strongly attenuates cryptococcal virulence in vivo. We propose that Skn7 (co-)regulates the fungal adaptive strategy, allowing intraphagocytic survival by conferring resistance to phagolysosomal killing in endothelial cells.

O-acetylation of cryptococcal capsular glucuronoxylomannan is essential for interference with neutrophil migration.

The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans has been shown to interfere with neutrophil migration. Although several receptors have been implied to mediate this process, the structural perspectives are unknown. Here, we assess the contribution of 6-O-acetylation and xylose substitution of the (1-->3)-alpha-d-mannan backbone of GXM, the variable structural features of GXM, to the interference with neutrophil migration. We compare chemically deacetylated GXM and acetyl- or xylose-deficient GXM from genetically modified strains with wild-type GXM in their ability to inhibit the different phases of neutrophil migration. Additionally, we verify the effects of de-O-acetylation on neutrophil migration in vivo. De-O-acetylation caused a dramatic reduction of the inhibitory capacity of GXM in the in vitro assays for neutrophil chemokinesis, rolling on E-selectin and firm adhesion to endothelium. Genetic removal of xylose only marginally reduced the ability of GXM to reduce firm adhesion. In vivo, chemical deacetylation of GXM significantly reduced its ability to interfere with neutrophil recruitment in a model of myocardial ischemia (65% reduction vs a nonsignificant reduction in tissue myeloperoxidase, respectively). Our findings indicate that 6-O-acetylated mannose of GXM is a crucial motive for the inhibition of neutrophil recruitment.

Intrathecal production and secretion of vascular endothelial growth factor during Cryptococcal Meningitis.

BACKGROUND: Patients with cryptococcal meningitis (CM) show elevated intracranial pressure (ICP) and blood-brain barrier (BBB) disruption in most cases. Elevated ICP is an important contributor to mortality. Vascular endothelial growth factor (VEGF) might be the mediator of BBB disruption during CM. METHODS: We measured VEGF levels in serum, plasma, and cerebrospinal fluid (CSF) of 95 patients and 63 control subjects, and we analyzed the required trigger and cellular source of VEGF secretion in vitro. RESULTS: Cryptococcus neoformans and its capsular antigens dose-dependently induced VEGF secretion by polymorphonuclear neutrophils, monocytes, and peripheral blood mononuclear cells (PBMCs). VEGF production by PBMCs induced by antigens strongly exceeded production by monocytes (P<.001). The addition of major histocompatibility complex class II antibody inhibited this production of VEGF (P=.005). Confirming the in vitro data, patients with CM showed significantly elevated VEGF levels in CSF (P<.001), plasma (P=.028), and serum (P<.001), compared with healthy control subjects. Calculated VEGF indices demonstrated that VEGF was produced intrathecally. CONCLUSIONS: Our findings suggest that VEGF plays a role in the pathophysiology of CM. We propose that CD4(+) T lymphocytes--stimulated by monocytes acting as antigen-presenting cells--are the cells that produce VEGF in response to cryptococcal antigens.