First large-scale study of antimicrobial susceptibility data, and genetic resistance determinants, in Fusobacterium necrophorum highlighting the importance of continuing focused susceptibility trend surveillance.

OBJECTIVES: The objective of the study was to explore antimicrobial resistance gene determinant, and phenotypic antibiotic susceptibility, data for Fusobacterium necrophorum from a collection of UK strains. Antimicrobial resistance genes detected in publicly available assembled whole genome sequences were investigated for comparison. METHODS: Three hundred and eighty five F. necrophorum strains (1982-2019) were revived from cryovials (Prolab). Subsequent to sequencing (Illumina) and quality checking, 374 whole genomes were available for analysis. Genomes were interrogated, using BioNumerics (bioMérieux; v 8.1), for the presence of known antimicrobial resistance genes (ARGs). Agar dilution susceptibility results for 313 F. necrophorum isolates (2016-2021) were also examined. RESULTS: The phenotypic data for the 313 contemporary strains demonstrated potential resistance to penicillin in three isolates, using EUCAST v 11.0 breakpoints, and 73 (23%) strains using v 13.0 analysis. All strains were susceptible to multiple agents using v 11.0 guidance other than clindamycin (n = 2). Employing v 13.0 breakpoints, metronidazole (n = 3) and meropenem (n = 13) resistance were also detected. The tet(O), tet(M), tet(40), aph(3')-III, ant(6)-la and blaOXA-85 ARGs were present in publicly available genomes. tet(M), tet(32), erm(A) and erm(B) were found within the UK strains, with correspondingly raised clindamycin and tetracycline minimum inhibitory concentrations. CONCLUSIONS: Susceptibility to antibiotics recommended for the treatment of F. necrophorum infections should not be assumed. With evidence of potential ARG transmission from oral bacteria, and the detection of a transposon-mediated beta-lactamase resistance determinant in F. necrophorum, surveillance of both phenotypic and genotypic antimicrobial susceptibility trends must continue, and increase.

Substantial Intestinal Microbiota Differences Between Patients With Ulcerative Colitis From Ghana and Denmark.

Background: Ulcerative colitis (UC) is a relapsing nontransmural inflammatory disease that is restricted to the colon and is characterized by flare-ups of bloody diarrhea. In this study, we aimed to investigate intestinal bacterial diversity in healthy controls and patients with UC with and without active disease, from Ghana and Denmark. Methods: The study included 18 UC patients (9 with active and 9 with inactive disease) and 18 healthy controls from Ghana. In addition 16 UC patients from Denmark (8 UC with active and 8 UC with inactive disease) and 19 healthy controls from Denmark. Microbiota diversity analysis relied on sequencing of ribosomal small subunit genes. Purified genomic DNA was submitted to PCR using a primer set targeting prokaryotes and eukaryotes. The purified DNA was sequenced on the Illumina MiSeq system in a 2 × 250 bp set up (Illumina, San Diego, CA, USA). Blinded analysis of the taxonomy table was performed using BioNumerics-7.5 (Applied Maths NV, Sint-Martens-Latem, Belgium). Results: When analyzing the taxonomy data for prokaryotes, cluster and principal component analysis shows Danish healthy controls clustered together, but separate from healthy controls from Ghana, which also clustered together. The Shannon diversity index (SDI) for prokaryotes shows significant differences between Danish healthy controls and patients in comparison with the corresponding groups from Ghana (p = 0.0056). Significant increased abundance of Escherichia coli was detected in healthy controls from Ghana in comparison with healthy controls from Denmark. The SDI of the prokaryotes ranges between 0 and 3.1 in the Ghana study groups, while in the Danish study groups it ranges between 1.4 and 3.2, the difference is however not significant (p = 0.138). Our data show a significant increased abundance of eukaryotes species in the healthy control group from Ghana and Denmark in comparison with patient groups from Ghana and Denmark. Conclusion: Overall, healthy controls and patients with UC from Denmark have increased diversity of prokaryotes. Healthy controls from Denmark and Ghana have increased abundance of eukaryotes in comparison with UC patient groups from Denmark and Ghana.

WGS-Based Prediction and Analysis of Antimicrobial Resistance in Campylobacter jejuni Isolates From Israel.

Rapid developments in the field of whole genome sequencing (WGS) make in silico antimicrobial resistance (AMR) a target within reach. Campylobacter jejuni is a leading cause of foodborne infections in Israel with increasing rates of resistance. We applied WGS analysis to study the prevalence and genetic basis of AMR in 263 C. jejuni human and veterinary representative isolates retrieved from a national collection during 2003-2012. We evaluated the prediction of phenotypic AMR from genomic data. Genomes were screened by the NCBI AMRFinderPlus and the BioNumerics tools for acquired AMR genes and point mutations. The results were compared to phenotypic resistance determined by broth microdilution. The most prevalent resistant determinants were the multi-drug efflux transporter gene cmeABC (100%), the tetracycline resistance tet(O) gene (82.1%), the quinolone resistance gyrA T861 point mutation (75.7%), and the aadE streptomycin resistance gene. A variety of 12 known ß lactam resistance genes (blaOXA variants) were detected in 241 (92%) isolates, the most prevalent being blaOXA-193, blaOXA-461, and blaOXA-580 (56, 16, and 7%, respectively). Other aminoglycoside resistance genes and the macrolide resistance point mutation were rare (<1%). The overall correlation rate between WGS-based genotypic prediction and phenotypic resistance was 98.8%, sensitivity, specificity, positive, and negative predictive values being 98.0, 99.3, 99.1, and 98.5%, respectively. wgMLST-based phylogeny indicated a high level of clonality and clustering among the studied isolates. Closely related isolates that were part of a genetic cluster (single linkage distance ≤ 15 alleles) based on wgMLST phylogeny mostly shared a homogenous AMR determinant profile. This was observed in 18 of 20 (90.0%) clusters within clonal complex-21, suggesting clonal expansion of resistant isolates. Strong association to lineage was noted for the aadE gene and the various blaOXA genes. High resistance rates to tetracycline and quinolones and a low resistance rate to macrolides were detected among the Israeli C. jejuni isolates. While a high genotypic-phenotypic correlation was found, some resistance phenotypes could not be predicted by the presence of AMR determinants, and particularly not the level of resistance. WGS-based prediction of antimicrobial resistance in C. jejuni requires further optimization in order to integrate this approach in the routine workflow of public health laboratories for foodborne surveillance.

Genomic Epidemiology of Campylobacter jejuni Transmission in Israel.

Objectives: Campylobacter jejuni is responsible for 80% of Campylobacter infections in Israel, a country with a high incidence reaching 91/100,000 population. We studied the phylogeny, diversity and prevalence of virulence factors using whole genome sequencing (WGS) of a national sample of C. jejuni clinical, food, and animal isolates collected over a 10-year period (2003-2012). Methods: C. jejuni isolates (n = 263) were subject to WGS using Illumina sequencing (PE 250bpx2). Raw reads and de novo assemblies were analyzed with the BioNumerics whole genome MLST (wgMLST) pipeline. Reads were screened for 71 virulence genes by the SRST2 script. Allelic profiles were analyzed to create minimum spanning trees and allelic core distances were investigated to determine a reliable cutoff for strain determination. Results: wgMLST analysis of 263 C. jejuni isolates indicated significant diversity among the prevalent clonal complexes (CCs) with CC-21 and CC-353 being the most diverse, and CC-574 the most clonal. Within CC-21, sequence type (ST)-1359 created a separate clade. Human, poultry and bovine isolates clustered together across the different STs. Forty four percent of studied isolates were assigned to 29 genetic clusters. Temporal and geographical relatedness were found among the minority of clusters, while most phylogenetically associated cases appeared diffuse and unassociated epidemiologically. The majority of virulence factors were highly prevalent across the dataset and not associated with genotype, source of isolation or invasiveness. Conversely, all 13 genes associated with type VI secretion system (T6SS) were lineage-related and identified in only 18% of the isolates. T6SS was detected in 95.2% of ST-1359, a common type in Israel. Conclusions: wgMLST supported the assessment that poultry and cattle are likely food sources of infection in Israel. Substantial genetic clustering among C. jejuni isolates suggested multiple point source and diffuse outbreaks that were previously unreported in Israel. The high prevalence of T6SS among ST-1359 isolates is unique to Israel, and requires further investigation. This study exemplifies the importance of studying foodborne pathogens using advanced genomic approaches across the entire spectrum of One Health.

Application of MALDI-TOF MS for the subtyping of Arcobacter butzleri strains and comparison with their MLST and PFGE types.

For the first time, this study evaluated the use of MALDI-TOF as a typing tool for Arcobacter butzleri. A total of 104 A. butzleri strains isolated from different sources in an artisanal dairy plant in Italy were identified and typed using MALDI-TOF and compared with their multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) profiles found in previous studies. MALDI-TOF correctly identified all the isolates to species level. No clearly delineated clusters appeared on dendrograms based on either the complete spectra or the significant peaks, but nine clusters were defined using the cophenetic correlation. Interestingly, MALDI-TOF proved able to discriminate A. butzleri strains below species level, confirming its potential use for epidemiological surveys. As expected, the comparative analysis with PFGE and MLST showed that the discriminatory index was lower for MALDI-TOF but roughly comparable to sequence types and pulsotypes. MALDI-TOF appears to be a relatively low cost answer to the urgent need for more rapid, less expensive typing tools suitable for source attribution studies, readily allowing multiple typing methods to be combined. This study provides insights into MALDI-TOF as potential epidemiological tool. Its application in healthcare surveillance systems awaits further exploration to encourage interaction and convergence studies between primary care in humans and animal and food veterinary authorities as part of the One Health concept.

Low prevalence of human enteropathogenic Yersinia spp. in brown rats (Rattus norvegicus) in Flanders.

Brown rats (Rattus norvegicus) have been identified as potential carriers of Yersinia enterocolitica and Y. pseudotuberculosis, the etiological agents of yersiniosis, the third most reported bacterial zoonosis in Europe. Enteropathogenic Yersinia spp. are most often isolated from rats during yersiniosis cases in animals and humans, and from rats inhabiting farms and slaughterhouses. Information is however lacking regarding the extent to which rats act as carriers of these Yersinia spp.. In 2013, 1088 brown rats across Flanders, Belgium, were tested for the presence of Yersinia species by isolation method. Identification was performed using MALDI-TOF MS, PCR on chromosomal- and plasmid-borne virulence genes, biotyping and serotyping. Yersinia spp. were isolated from 38.4% of the rats. Of these, 53.4% were designated Y. enterocolitica, 0.7% Y. pseudotuberculosis and 49.0% other Yersinia species. Two Y. enterocolitica possessing the virF-, ail- and ystA-gene were isolated. Additionally, the ystB-gene was identified in 94.1% of the other Y. enterocolitica isolates, suggestive for biotype 1A. Three of these latter isolates simultaneously possessed the ail-virulence gene. Significantly more Y. enterocolitica were isolated during winter and spring compared to summer. Based on our findings we can conclude that brown rats are frequent carriers for various Yersinia spp., including Y. pseudotuberculosis and (human pathogenic) Y. enterocolitica which are more often isolated during winter and spring.

Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs.

The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two-six strains, ≥seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.-max.) number of different strains per batch were 7 (1-13). Batches with two-six strains or more than six strains had more severe Mycoplasma-like lesions (P = 0.064 and P = 0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P = 0.33 and OR: 2.08, P = 0.012, respectively], and fissures (OR = 1.35, P = 0.094 and OR = 1.70, P = 0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs.

The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Based Typing: Employment of Bioinformatics in a Multicenter Study.

BACKGROUND: The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) typing data has not yet been explored. The aim of this study is to compare typing data from multiple centers employing bioinformatics using bacterial strains from two past outbreaks and non-related strains. MATERIAL/METHODS: Participants received twelve extended spectrum betalactamase-producing E. coli isolates and followed the same standard operating procedure (SOP) including a full-protein extraction protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Germany). Raw data from each laboratory allowed calculating the technical and biological reproducibility between centers using BioNumerics (Applied Maths NV, Belgium). RESULTS: Technical and biological reproducibility ranged between 96.8-99.4% and 47.6-94.4%, respectively. The inter-center reproducibility showed a comparable clustering among identical isolates. Principal component analysis indicated a higher tendency to cluster within the same center. Therefore, we used a discriminant analysis, which completely separated the clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster. CONCLUSIONS: Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different centers. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable.

Epidemic Achromobacter xylosoxidans strain among Belgian cystic fibrosis patients and review of literature.

BACKGROUND: Achromobacter xylosoxidans is increasingly being recognized as an emerging pathogen in cystic fibrosis. Recent severe infections with A. xylosoxidans in some of our cystic fibrosis (CF) patients led to a re-evaluation of the epidemiology of CF-associated A. xylosoxidans infections in two Belgian reference centres (Antwerp and Ghent). Several of these patients also stayed at the Rehabilitation Centre De Haan (RHC). In total, 59 A. xylosoxidans isolates from 31 patients (including 26 CF patients), collected between 2001 and 2014, were studied. We evaluated Matrix Assisted Laser Desorption Ionisation -Time of Flight mass spectrometry (MALDI-TOF) as an alternative for McRAPD typing. RESULTS: Both typing approaches established the presence of a major cluster, comprising isolates, all from 21 CF patients, including from two patients sampled when staying at the RHC a decade ago. This major cluster was the same as the cluster established already a decade ago at the RHC. A minor cluster consisted of 13 isolates from miscellaneous origin. A further seven isolates, including one from a non-CF patient who had stayed recently at the RHC, were singletons. CONCLUSIONS: Typing results of both methods were similar, indicating transmission of a single clone of A. xylosoxidans among several CF patients from at least two reference centres. Isolates of the same clone were already observed at the RHC, a decade ago. It is difficult to establish to what extent the RHC is the source of transmission, because the epidemic strain was already present when the first epidemiological study in the RHC was carried out. This study also documents the applicability of MALDI-TOF for typing of strains within the species A. xylosoxidans and the need to use the dynamic cutoff algorithm of the BioNumerics® software for correct clustering of the fingerprints.

Use of MALDI-TOF mass spectrometry and a custom database to characterize bacteria indigenous to a unique cave environment (Kartchner Caverns, AZ, USA).

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine.

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn(®)MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p<0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p<0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.

Vaccination reduces macrophage infiltration in bronchus-associated lymphoid tissue in pigs infected with a highly virulent Mycoplasma hyopneumoniae strain.

BACKGROUND: Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia and is responsible for significant economic losses to the pig industry. To better understand the mode of action of a commercial, adjuvanted, inactivated whole cell vaccine and the influence of diversity on the efficacy of vaccination, we investigated samples from vaccinated and non-vaccinated pigs experimentally infected with either a low (LV) or a highly virulent (HV) M. hyopneumoniae strain. Non-vaccinated and sham-infected control groups were included. Lung tissue samples collected at 4 and 8 weeks post infection (PI) were immunohistochemically tested for the presence of T-lymphocytes, B-lymphocytes and macrophages in the bronchus-associated lymphoid tissue (BALT). The number of M. hyopneumoniae organisms in bronchoalveolar lavage (BAL) fluid was determined using quantitative PCR at 4 and 8 weeks PI. Serum antibodies against M. hyopneumoniae were determined at 0, 2, 4, 6 and 8 weeks PI. RESULTS: The immunostaining revealed a lower density of macrophages in the BALT of the vaccinated groups compared to the non-vaccinated groups. The highest number of M. hyopneumoniae organisms in the BAL fluid was measured at 4 weeks PI for the HV strain and at 8 weeks PI for the LV strain. Vaccination reduced the number of organisms non-significantly, though for the HV strain the reduction was clinically more relevant than for the LV strain. At the level of the individual pigs, a higher lung lesion score was associated with more M. hyopneumoniae organisms in the lungs and a higher density of the investigated immune cells in the BALT. CONCLUSIONS: In conclusion, the infiltration of macrophages after infection with M. hyopneumoniae is reduced by vaccination. The M. hyopneumoniae replication in the lungs is also reduced in vaccinated pigs, though the HV strain is inhibited more than the LV strain.

Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous Mycoplasma hyopneumoniae strains.

BACKGROUND: Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in pigs. Although vaccination is an important control tool, the results observed under field conditions are variable. This may be due to antigenic differences between the strains circulating in pig herds and the vaccine strain. This study compared the protective efficacy of four bacterins against challenge infection with a highly virulent field strain of M. hyopneumoniae. Seventy eight, one-week old piglets were randomly assigned to five treatment groups (A, B, C, D, E), 14 piglets each, and a negative control group (F) consisting of 8 piglets. All pigs were injected at 1 (D7) and 4 weeks of age (D28), with 2 ml of either a placebo or a bacterin based on selected M. hyopneumoniae strains, namely A (F7.2C), B (F20.1L), C (B2V1W20 1A-F), D (J strain), E (placebo; positive control), F (placebo; negative control). At D56, all pigs except those of group F were challenged intratracheally with 7 ml culture medium containing 107 CCU/ml of M. hyopneumoniae strain F7.2C. All pigs were euthanized and necropsied at D84. The severity of coughing and pneumonia lesions were the main parameters. Immunofluorescence (IF) testing, nested PCR testing of bronchoalveolar lavage (BAL) fluid and serology for M. hyopneumoniae were also performed. RESULTS: The different bacterins only slightly improved clinical symptoms (average 0.38 in vaccinated groups vs. 0.45 in group E) and histopathological lung lesions (average 3.20 in vaccinated groups vs. 3.45 in group E), but did not improve macroscopic lung lesions (score 4.30 vs. 4.03 in group E). None of the vaccines was significantly and/or consistently better or worse than the other ones. All bacterins evoked a serological response in the vaccinated animals. All pigs, except those from group F, were positive with nPCR in BAL fluid at D84. CONCLUSION: The bacterins did not induce a clear overall protection against challenge infection, and there were no significant differences in protective efficacy between bacterins containing homologous and heterologous M. hyopneumoniae strains. Further research is necessary to better characterize the antigens involved in protection and to elucidate the protective immunity responses following M. hyopneumoniae vaccination and/or infection.

A longitudinal study of the diversity and dynamics of Mycoplasma hyopneumoniae infections in pig herds.

A longitudinal study was carried out to investigate the diversity and persistence of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains in four infected pig herds. In each herd, 20 pigs were randomly selected and blood and/or bronchoalveolar lavage (BAL) fluid was collected at 6, 10, 14 and 26 weeks of age. In the BAL fluid, quantitative PCR and MLVA (multiple-locus variable number of tandem repeats (VNTR) analysis) testing were performed for detection and typing of M. hyopneumoniae strains, respectively. At 26 weeks of age, the prevalence and severity of lung lesions were recorded at slaughter (minimum 50 pigs belonging to the same batch as the investigated pigs). The percentage of pigs testing positive on qPCR increased from 35% at 6 weeks to 96% at 26 weeks of age. With MLVA testing, positive pigs were found from 14 weeks onwards. Within each herd, only one distinct strain was detected, although clonal variants were identified in two herds. In three of the herds, the strain remained present until slaughter age. The percentage of pigs with Mycoplasma-like lesions ranged from 38% to 98%, and the average pneumonia score ranged from 1.7 to 11.9, respectively. The present field study documented that within a herd, mainly one distinct M. hyopneumoniae strain was present that persisted in the same animals for at least 12 weeks. This implies that the immune response of the animals following infection is not able to rapidly clear the infection from the respiratory tract.

L-beta-ODAP alters mitochondrial Ca2+ handling as an early event in excitotoxicity.

The neurotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (L-beta-ODAP) is an L-glutamate analogue at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors in neurons and therefore acts as an excitotoxic substance. Chronic exposure to L-beta-ODAP present in Lathyrus sativus L. (L. sativus) seeds is proposed as the cause of the neurodegenerative disease neurolathyrism, but the mechanism of its action has not been conclusively identified. A key factor in excitotoxic neuronal cell death is a disturbance of the intracellular Ca2+ homeostasis, including changes in the capacity of intracellular Ca2+ stores like the endoplasmic reticulum (ER) or mitochondria. In this study, aequorin and other Ca2+ indicators were used in N2a neuroblastoma cells to investigate alterations of cellular Ca2+ handling after 24 h exposure to L-beta-ODAP. Our data demonstrate increased mitochondrial Ca2+ loading and hyperpolarization of the mitochondrial membrane potential (Psi(m)), which was specific for L-beta-ODAP and not observed with L-glutamate. We conclude that L-beta-ODAP disturbs the ER-mitochondrial Ca2+ signaling axis and thereby renders the cells more vulnerable to its excitotoxic effects that ultimately will lead to cell death.