Fourier-transform infrared spectroscopy for typing of vancomycin-resistant Enterococcus faecium: performance analysis and outbreak investigation.

Vancomycin-resistant Enterococci, mainly Enterococcus faecium (VREfm), are causing nosocomial infections and outbreaks. Bacterial typing methods are used to assist in outbreak investigations. Most of them, especially genotypic methods like multi-locus sequence typing (MLST), whole genome sequencing (WGS), or pulsed-field gel electrophoresis, are quite expensive and time-consuming. Fourier-transform infrared (FT-IR) spectroscopy assesses the biochemical composition of bacteria, such as carboxyl groups in polysaccharides. It is an affordable technique and has a faster turnaround time. Thus, the aim of this study was to evaluate FT-IR spectroscopy for VREfm outbreak investigations. Basic performance requirements like reproducibility and the effects of incubation time were assessed in distinct sample sets. After determining a FT-IR spectroscopy cut-off range, the clustering agreement between FT-IR and WGS within a retrospective (n: 92 isolates) and a prospective outbreak (n: 15 isolates) was investigated. For WGS an average nucleotide identity (ANI) cut-off score of 0.999 was used. Basic performance analysis showed reproducible results. Moreover, FT-IR spectroscopy readouts showed a high agreement with WGS-ANI analysis in clinical outbreak investigations (V-measure 0.772 for the retrospective and 1.000 for the prospective outbreak). FT-IR spectroscopy had a higher discriminatory power than MLST in the outbreak investigations. After determining cut-off values to achieve optimal resolution, FT-IR spectroscopy is a promising technique to assist in outbreak investigation as an affordable, easy-to-use tool with a turnaround time of less than one day. IMPORTANCE Vancomycin-resistant Enterococci, mainly Enterococcus faecium (VREfm), are a frequent cause of nosocomial outbreaks. Several bacterial typing methods are used to track transmissions and investigate outbreaks, whereby genome-based techniques are used as a gold standard. Current methods are either expensive, time-consuming, or both. Additionally, often, specifically trained staff needs to be available. This study provides insight into the use of Fourier-transform infrared (FT-IR) spectroscopy, an affordable, easy-to-use tool with a short turnaround time as a typing method for VREfm. By assessing clinical samples, this work demonstrates promising results for species discrimination and reproducibility. FT-IR spectrosopy shows a high level of agreement in the analysis of VREfm outbreaks in comparison with whole genome sequencing-based methods.

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model.

OBJECTIVES: Streptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall-active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model. METHODS: We investigated the effect of IVIG on the activity of the virulence factors streptolysin O (SLO), streptodornase 1 (Sda1), S. pyogenes cell envelope protease and streptococcal pyrogenic exotoxin B in vitro and ex vivo in patient sera. Additionally, we assessed the influence of IVIG on the clinical outcome in a murine NF model. RESULTS: In vitro, IVIG inhibited various streptococcal virulence factors. Further, IVIG treatment of group A Streptococcus-infected mice led to a reduced skin lesion size (median (interquartile range) day 3 intraperitoneal administration: 12 mm2 (9-14.5) vs. 4 mm2 (0.8-10.5), subcutaneous: 10.3 mm2 (6.9-18.6) vs. 0.5 mm2 (0.1-6.8)) and lower SLO activity. After treatment with IVIG, patient sera showed an elevated titre of specific SLO (7/9) and Sda1 (5/9) antibodies, reducing SLO and Sda1 activity. CONCLUSIONS: The clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.

Chloroquine enhances the antimycobacterial activity of isoniazid and pyrazinamide by reversing inflammation-induced macrophage efflux.

Mycobacterium tuberculosis (MTB) is notorious for persisting within host macrophages. Efflux pumps decrease intracellular drug levels, thus fostering persistence of MTB during therapy. Isoniazid (INH) and pyrazinamide (PZA) are substrates of the efflux pump breast cancer resistance protein-1 (BCRP-1), which is inhibited by chloroquine (CQ). In this study, BCRP-1 was found to be expressed on macrophages of human origin and on foamy giant cells at the site of MTB infection. In the current in vitro study, interferon-gamma (IFNγ) increased the expression of BCRP-1 in macrophages derived from the human monocytic leukaemia cell line THP-1. Using a BCRP-1-specific fluorescent dye and radioactively labelled INH, it was demonstrated that efflux from macrophages increased upon activation with IFNγ. CQ was able to inhibit active efflux and augmented the intracellular concentrations both of INH and the dye. In agreement, CQ and specific inhibition of BCRP-1 increased the antimycobacterial activity of INH against intracellular MTB. Although PZA behaved differently, CQ had comparable advantageous effects on the intracellular pharmacokinetics and activity of PZA. The adjunctive effects of CQ on intracellular killing of MTB were measurable at concentrations achievable in humans at approved therapeutic doses. Therefore, CQ, a widely used and worldwide available drug, may potentiate the efficacy of standard MTB therapy against bacteria in the intracellular compartment.

Development of a multiplex PCR assay for Photobacterium damselae subsp. piscicida identification in fish samples.

A multiplex polymerase chain reaction protocol for the detection of Photobacterium damselae and subspecies piscicida and damselae discrimination, with internal amplification control, was developed. Assay specificity was assessed by testing 19 target and 25 non-target pure cultures. The detection limit was 500 fg, corresponding to 100 genome equivalents. The optimized protocol was also prevalidated with spleen, kidney and blood samples from infected and uninfected sea bass, without any culture step, and it can be proposed as a valid alternative to culture standard methods for the rapid and specific diagnosis of photobacteriosis in fish.

Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida.

Abstract Cobalamin (vitamin B(12)) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B(12). A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B(12)-binding protein precursor of P. profundum, 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida, but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida, the recombinant protein was expressed with a C-terminal His(6)-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 +/- 2 microm.

Loss of pericentromeric DNA methylation pattern in human glioblastoma is associated with altered DNA methyltransferases expression and involves the stem cell compartment.

Cancer is generally characterized by loss of CG dinucleotides methylation resulting in a global hypomethylation and the consequent genomic instability. The major contribution to the general decreased methylation levels seems to be due to demethylation of heterochromatin repetitive DNA sequences. In human immunodeficiency, centromeric instability and facial anomalies syndrome, demethylation of pericentromeric satellite 2 DNA sequences has been correlated to functional mutations of the de novo DNA methyltransferase 3b (DNMT3b), but the mechanism responsible for the hypomethylated status in tumors is poorly known. Here, we report that human glioblastoma is affected by strong hypomethylation of satellite 2 pericentromeric sequences that involves the stem cell compartment. Concomitantly with the integrity of the DNMTs coding sequences, we report aberrations in DNA methyltrasferases expression showing upregulation of the DNA methyltransferase 1 (DNMT1) and downregulation of the de novo DNA methyltransferase 3a (DNMT3a). Moreover, we show that DNMT3a is the major de novo methyltransferase expressed in normal neural progenitor cells (NPCs) and its forced re-expression is sufficient to partially recover the methylation levels of satellite 2 repeats in glioblastoma cell lines. Thus, we speculate that DNMT3a decreased expression may be involved in the early post-natal inheritance of an epigenetically altered NPC population that could be responsible for glioblastoma development later in adult life.

Pharmacogenetic profiling in patients with advanced colorectal cancer treated with first-line FOLFIRI chemotherapy.

The primary end point of the study was the analysis of associations between polymorphisms with putative influence on 5-fluorouracil/irinotecan activity and progression-free survival (PFS) of patients with advanced colorectal cancer treated with first-line FOLFIRI chemotherapy. Peripheral blood samples from 146 prospectively enrolled patients were used for genotyping polymorphisms in thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), excision repair cross-complementation group-1 (ERCC 1) xeroderma pigmentosum group-D (XPD), X-ray cross-complementing-1 (XRCC 1), X-ray cross-complementing-3 (XRCC 3) and uridine diphosphate-glucuronosyltransferases-A1 (UGT1 A1). TS 3'-UTR 6+/6+ and XRCC3-241 C/C genotypes were associated with adverse PFS. Hazard ratio for PFS achieved 2.89 (95% confidence interval=1.56-5.80; P=0.002) in 30 patients (20%) with both risk genotypes. Risk for Grade III-IV neutropenia was significantly associated with UGT1A1*28 7/7 genotype. These promising findings deserve further investigations and their validation in independent prospective studies.

Structure of the 5' region of the human hexokinase type I (HKI) gene and identification of an additional testis-specific HKI mRNA.

We previously reported the structure of the human hexokinase type I (HKI) gene and provided direct evidence of an alternative red blood cell-specific exon 1 located in the 5' flanking region of the gene. Three unique HKI mRNA species have also been described in human spermatogenic cells. These mRNAs contain a testis-specific sequence not present in somatic cell HKI, but lack the sequence for the porin-binding domain necessary for HKI to bind to porin on the outer mitochondrial membrane. The present study reports a new mRNA isoform, hHKI-td, isolated from human sperm. hHKI-td mRNA contains both a testis-specific sequence at the 5' end common to the three other mRNA isoforms and an additional unique sequence. Screening of a cosmid library and analysis of the cosmids containing the HKI gene revealed that testis-specific sequences are encoded by six different exons. Five of these exons are located upstream from the somatic exon 1 (5.6-30 kb) and one within intron 1. This study shows that a single human HKI gene spanning at least 100 kb encodes multiple transcripts that are generated by alternative splicing of different 5' exons. Testis-specific transcripts are probably produced by a separate promoter that induces the expression of the HKI gene in spermatogenic cells.

Structure of the human hexokinase type I gene and nucleotide sequence of the 5' flanking region.

This study reports the precise intron/exon boundaries and intron/exon composition of the human hexokinase type I gene. A yeast artificial chromosome containing the hexokinase type I gene was isolated from the yeast artificial chromosome library of the Centre d'Etude du Polymorphisme Humaine. A cosmid sublibrary was created and direct sequencing of the individual cosmids was used to provide the exon/intron organization. The human hexokinase type I gene was found to be composed of 18 exons ranging in size from 63 to 305 bp. Intron 1 is at least 15 kb in length, whereas intron 2 spans at least 10 kb. Overall, the length of the 17 introns ranges from 104 to greater than 15 kb. The entire coding region is contained in at least 75 kb of the gene. The structure of the gene reveals a remarkable conservation of the size of the exons compared with glucokinase and hexokinase type II. Isolation of the 5' flanking region of the gene revealed a 75-90% identity with the rat sequence. Direct evidence of an alternative red-blood-cell-specific exon 1 located upstream of the 5' flanking region of the gene is also provided.