Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant.

Beta-lactam resistant clinical isolates of Streptococcus pneumoniae contain altered penicillin-binding protein (PBP) genes and occasionally an altered murM, presumably products of interspecies gene transfer. MurM and MurN are responsible for the synthesis of branched lipid II, substrate for the PBP catalyzed transpeptidation reaction. Here we used the high-level beta-lactam resistant S. oralis Uo5 as donor in transformation experiments with the sensitive laboratory strain S. pneumoniae R6 as recipient. Surprisingly, piperacillin-resistant transformants contained no alterations in PBP genes but carried murEUo5 encoding the UDP-N-acetylmuramyl tripeptide synthetase. Codons 83-183 of murEUo5 were sufficient to confer the resistance phenotype. Moreover, the promoter of murEUo5 , which drives a twofold higher expression compared to that of S. pneumoniae R6, could also confer increased resistance. Multiple independent transformations produced S. pneumoniae R6 derivatives containing murEUo5 , pbp2xUo5 , pbp1aUo5 and pbp2bUo5 , but not murMUo5 sequences; however, the resistance level of the donor strain could not be reached. S. oralis Uo5 harbors an unusual murM, and murN is absent. Accordingly, the peptidoglycan of S. oralis Uo5 contained interpeptide bridges with one L-Ala residue only. The data suggest that resistance in S. oralis Uo5 is based on a complex interplay of distinct PBPs and other enzymes involved in peptidoglycan biosynthesis.

Procalcitonin as a biomarker in equine chronic pneumopathies.

BACKGROUND: Procalcitonin (PCT), a precursor protein of the hormone calcitonin, is a sensitive inflammatory marker in human medicine, which is primarily used for diagnosis of bacterial sepsis, but is also useful in diagnosis of exacerbation of asthma and COPD. In this study, PCT was evaluated as a potential biomarker for different chronic pneumopathies in the horse using an equine specific ELISA in comparison to established clinical markers and different interleukins. Sixty-four horses were classified as free of respiratory disease, recurrent airway obstruction (RAO), inflammatory airway disease (IAD) or chronic interstitial pneumopathy (CIP) using a scoring system. PCT concentrations were measured in plasma (n = 17) and in the cell-free supernatant of bronchoalveolar lavage (n = 64). PCT concentrations were correlated to interleukins IL-1ß and IL-6 in BALF, clinical findings and BALF cytology. RESULTS: The median PCT concentrations in plasma were increased in respiratory disease (174.46 ng/ml, n = 7) compared to controls (13.94 ng/ml, n = 10, P = 0.05) and correlated to PCT in BALF supernatant (rs = 0.48). Compared to controls (5.49 ng/ml, n = 15), median PCT concentrations in BALF supernatant correlated to the overall clinical score (rs = 0.32, P = 0.007) and were significantly increased in RAO (13.40 ng/ml, n = 21) and IAD (16.89 ng/ml, n = 16), while no differences were found for CIP (12.02 ng/ml, n = 12). No significant increases were found for IL-1 and IL-6 between controls and respiratory disease in general as well as different disease groups. CONCLUSIONS: Although some correlations were found between PCT in plasma, BALF supernatant and clinical scores, PCT in BALF does not seem to be a superior marker compared to established clinical markers. PCT in plasma seems to be more promising and a greater number of samples should be evaluated in further studies.

A new ELISA for the quantification of equine procalcitonin in plasma as potential inflammation biomarker in horses.

In human medicine, procalcitonin (PCT) is a very common and well-established biomarker for sepsis. Even though sepsis is also a leading cause of death in foals and adult horses, up to now, no data about the role of equine PCT in septic horses has been available. Based on monoclonal antibodies targeted against human PCT, we report here the development of a sandwich ELISA for the quantification of equine PCT in equine plasma samples. The ELISA was characterized for intra- and interassay variance and a working range from 25 to 1,000 ng mL(-1) was defined as within this range; both intra- and interassay variances were below 15 %. The target recovery ranged between 73 and 106 %. The ELISA was used to determine the equine PCT concentration in 24 healthy and 5 septic horses to show the potential for clinical evaluation of equine PCT. Significantly different (P = 0.0006) mean equine PCT concentrations were found for the healthy control group and the sepsis group (47 and 8,450 ng mL(-1)).

Analysis of N-acylhomoserine lactone dynamics in continuous cultures of Pseudomonas putida IsoF by use of ELISA and UHPLC/qTOF-MS-derived measurements and mathematical models.

In this interdisciplinary approach, the dynamics of production and degradation of the quorum sensing signal 3-oxo-decanoylhomoserine lactone were studied for continuous cultures of Pseudomonas putida IsoF. The signal concentrations were quantified over time by use of monoclonal antibodies and ELISA. The results were verified by use of ultra-high-performance liquid chromatography. By use of a mathematical model we derived quantitative values for non-induced and induced signal production rate per cell. It is worthy of note that we found rather constant values for different rates of dilution in the chemostat, and the values seemed close to those reported for batch cultures. Thus, the quorum-sensing system in P. putida IsoF is remarkably stable under different environmental conditions. In all chemostat experiments, the signal concentration decreased strongly after a peak, because emerging lactonase activity led to a lower concentration under steady-state conditions. This lactonase activity probably is quorum sensing-regulated. The potential ecological implication of such unique regulation is discussed.

The European Bioanalysis Forum recommendation on establishing appropriate drug tolerance levels in antidrug antibody assays.

The European Bioanalysis Forum, in collaboration with several key industry stakeholders, has recently led discussions that address international immunogenicity guidance documents, specifically the three tier approach for immunogenicity testing strategies, after more than 20 years of experience with biotherapeutics. As part of this, the strategy and methods used to assess drug tolerance across all immunogenicity assays are challenged, emphasizing that bioanalytical scientists need to consider the context-of-use of each assay. Here, recommendations for drug tolerance assessments, driven by strong scientific rationale and subject to reevaluation as needed, are provided. This includes carefully considering the drug and positive control concentrations considered to be appropriate and which tiers are most relevant for performing drug tolerance assessments.

Streptococcus pneumoniae R6 interspecies transformation: genetic analysis of penicillin resistance determinants and genome-wide recombination events.

Interspecies gene transfer has been implicated as the major driving force for the evolution of penicillin resistance in Streptococcus pneumoniae. Genomic alterations of S. pneumoniae R6 introduced during four successive transformations with DNA of the high-level penicillin-resistant Streptococcus mitis B6 with beta-lactam selection have now been determined and the contribution of genes to high resistance levels was analysed genetically. Essential for high level resistance to penicillins of the transformant CCCB was the combination of murM(B) (6) and the 3' region of pbp2b(B) (6) . Sequences of both genes were detected in clinical isolates of S. pneumoniae, confirming the participation of S. mitis in the global gene pool of beta-lactam resistance determinants. The S. mitis PBP1b gene which contains an authentic stop codon within the transpeptidase domain is now shown to contribute only marginal to resistance, but it is possible that the presence of its transglycosylase domain is important in the context of cognate PBPs. The genome sequence of CCCB revealed 36 recombination events, including deletion and acquisition of genes and repeat elements. A total of 78 genes were affected representing 67 kb or 3.3% of the genome, documenting extensive alterations scattered throughout the genome.

Combination of crossflow ultrafiltration, monolithic affinity filtration, and quantitative reverse transcriptase PCR for rapid concentration and quantification of model viruses in water.

We present a rapid and effective adsorption-elution method based on monolithic affinity filtration (MAF) for the concentration and purification of waterborne viruses. The MAF column consists of a hydrolyzed macroporous epoxy-based polymer. High recoveries were achieved by columns for the bacterial virus (bacteriophage) MS2 110 (±19)%, as model organism, as well as for human adenoviruses 42.4 (±3.4)% and murine noroviruses 42.6 (±1.9)%. This new concentration and purification method was combined with crossflow ultrafiltration (CUF). Because of the adsorption of the examined viruses to the macroporous surface of the MAF column at pH 3, concentrated matrix components by CUF can be removed. Bacteriophages MS2 were spiked in tap water and concentrated with the new CUF-MAF concentration method by a volumetric factor of 10(4) within 33 min. Furthermore, the detection limit for quantification of bacteriophage MS2 by quantitative reverse transcriptase PCR (qRT-PCR) could be improved from 79.47 to 0.0056 GU mL(-1) by a factor of 1.4 × 10(4). In a first study, we have shown that this method could also be applied for river water containing naturally MS2 and MS2-like phages.

Development of antibody-labelled superparamagnetic nanoparticles for the visualisation of benzo[a]pyrene in porous media with magnetic resonance imaging.

Biogeochemical interfaces in soil are dynamic in the spatial and temporal domain and require advanced visualisation and quantification tools to link in vitro experiments with natural systems. This study presents the development, characterization and application of functional nanoparticles coated with monoclonal antibodies to visualise the distribution of benzo[a]pyrene in porous media using magnetic resonance imaging. The labelled particles are 450 nm in diameter and interact with benzo[a]pyrene covalently bound to silanized silica gel. They did not bind to benzo[a]pyrene adsorbed to plain silica gel. Although unspecific filtration was low, washing steps are required for visualisation. The ability to visualise benzo[a]pyrene is inversely correlated to the heterogeneity of the soil materials. There are access restrictions to narrow pore spaces which allow the visualisation of only those pathways which are also accessible to bacteria and hydrocolloids. The production of the particles is applicable to other antibodies which extends the range of potential target contaminants.

Genome of Streptococcus oralis strain Uo5.

Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci, which includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple-antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci and their potential to engage in interspecies gene transfer.

Characterization of an interstitial 4q32 deletion in a patient with mental retardation and a complex chromosome rearrangement.

Interstitial deletions of chromosome band 4q32 are rare. We report on a 22-year-old female patient with a de novo interstitial deletion of chromosome 4q32 and a balanced translocation t(2;5)(p21;q12.1). Clinical problems of the patient comprised mild to moderate mental retardation, psychosis, obesity, broad nasal root, sparse lateral eyebrows, thin upper lip, short philtrum, micrognathia, and strabismus. Analysis by whole genome array CGH using an Agilent 244K oligonucleotide array and subsequent FISH using BAC clones from the 4q32 region revealed an unexpectedly complex rearrangement comprising a deletion of approximately 10 Mb in 4q32.1q32.3 and the insertion of two small fragments of 0.8 and 0.11 Mb originating from the derivative chromosome 4q32 into derivative chromosome 5q. The breakpoints of the t(2;5) translocation were mapped by BAC-FISH; no genes were disrupted by these breakpoints. The deleted interval in 4q32 harbored more than 30 genes, and haploinsufficiency of one or several of these genes is likely to have caused the clinical problems of the patient. Candidate genes for cognitive defects are GRIA2, GLRB, NPY1R, and NPY5R. In conclusion, this patient increases our knowledge about the phenotypic consequences of interstitial 4q32 deletions. Reports of patients with overlapping deletions will be needed to elucidate the role of individual genes and to establish genotype-phenotype correlations.

Efficient hybridoma screening technique using capture antibody based microarrays.

The hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs), particularly if the target of the antibody is a low-molecular weight analyte. This work presents an advanced screening method that makes use of antibody microarrays generated by contact printing of the hybridoma cell supernatant samples on glass chips initially coated with capture antibodies. The noncompetitive immunoassay is based on the specific binding of an analyte-horseradish peroxidase conjugate and is performed in an automated fashion using a chemiluminescence readout system. Compared to the standard ELISA screening, the work load is reduced due to a higher degree of automation. The quality of the generated data is comparable to data generated by a previously optimized microplate-based immunoassay method. Among a reference set of 373 hybridoma cell supernatant samples, three out of four high-affinity MAbs were identified as true positive, whereas none of the samples were detected as a false positive.

Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient.

Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37 months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S. pneumoniae strains except for pbp1a of a Romanian S. pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S. pneumoniae to S. mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S. pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCEStreptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor.

Draft Genome Sequences of Two Streptococcus pneumoniae Serotype 19A Sequence Type 226 Clinical Isolates from Hungary, Hu17 with High-Level Beta-Lactam Resistance and Hu15 of a Penicillin-Sensitive Phenotype.

The draft genome sequences of two multiple-antibiotic-resistant Streptococcus pneumoniae isolates from Hungary, Hu15 and Hu17, are reported here. Strain Hu15 is penicillin susceptible, whereas Hu17 is a high-level-penicillin-resistant strain. Both isolates belong to the serotype 19A sequence type 226, a single-locus variant (in the ddl locus) of the Hungary19A-6 clone.

Highly Variable Streptococcus oralis Strains Are Common among Viridans Streptococci Isolated from Primates.

Viridans streptococci were obtained from primates (great apes, rhesus monkeys, and ring-tailed lemurs) held in captivity, as well as from free-living animals (chimpanzees and lemurs) for whom contact with humans is highly restricted. Isolates represented a variety of viridans streptococci, including unknown species. Streptococcus oralis was frequently isolated from samples from great apes. Genotypic methods revealed that most of the strains clustered on separate lineages outside the main cluster of human S. oralis strains. This suggests that S. oralis is part of the commensal flora in higher primates and evolved prior to humans. Many genes described as virulence factors in Streptococcus pneumoniae were present also in other viridans streptococcal genomes. Unlike in S. pneumoniae, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) gene clusters were common among viridans streptococci, and many S. oralis strains were type PI-2 (pilus islet 2) variants. S. oralis displayed a remarkable diversity of genes involved in the biosynthesis of peptidoglycan (penicillin-binding proteins and MurMN) and choline-containing teichoic acid. The small noncoding cia-dependent small RNAs (csRNAs) controlled by the response regulator CiaR might contribute to the genomic diversity, since we observed novel genomic islands between duplicated csRNAs, variably present in some isolates. All S. oralis genomes contained a ß-N-acetyl-hexosaminidase gene absent in S. pneumoniae, which in contrast frequently harbors the neuraminidases NanB/C, which are absent in S. oralis. The identification of S. oralis-specific genes will help us to understand their adaptation to diverse habitats. IMPORTANCE Streptococcus pneumoniae is a rare example of a human-pathogenic bacterium among viridans streptococci, which consist of commensal symbionts, such as the close relatives Streptococcus mitis and S. oralis. We have shown that S. oralis can frequently be isolated from primates and a variety of other viridans streptococci as well. Genes and genomic islands which are known pneumococcal virulence factors are present in S. oralis and S. mitis, documenting the widespread occurrence of these compounds, which encode surface and secreted proteins. The frequent occurrence of CRISP-Cas gene clusters and a surprising variation of a set of small noncoding RNAs are factors to be considered in future research to further our understanding of mechanisms involved in the genomic diversity driven by horizontal gene transfer among viridans streptococci.

[A new model of comprehensive data linkage--evaluation of its application in femoral neck fracture]. / Ein neues Modell der sektorübergreifenden Datenzusammenführung und Evaluation am Beispiel der Schenkelhalsfraktur.

Aim of the project was a comprehensive assessment of short- and middle-term outcome of femoral neck fracture by linkage and analysis of available data from routine care. For this purpose, a generic model of data linkage was developed, agreed with the data security officer, and practically applied. Included were all patients of the AOK Westphalia-Lippe, who were treated in a general or trauma surgery hospital in 1995/1999 for a femoral neck fracture (ICD-9: 820). For these patients, the linkage was based on the following sources: the data regarding the initial hospital stay were provided by the office of quality assurance of the chamber of physicians of Westphalia-Lippe; the administrative data were provided by the AOKWestphalia-Lippe; and the data evaluating the nursing needs were obtained from the Medical Services of the Health Insurance of Westphalia-Lippe (MDK). This paper presents the model of data linkage and describes its practical implementation; it also presents medical data demonstrating that femoral neck fractures are associated with high mortality and increase of nursing needs in the course of disease. The benefit of the new model is manifold and can be easily extended to other clinical questions.

Total internal reflection (TIRF)-based quantification of procalcitonin for sepsis diagnosis--a point-of-care testing application.

A new, highly sensitive fluorescence immunoassay for a TIRF (total internal reflection)-based point-of-care testing (POCT) device was developed for the detection of procalcitonin (PCT), a specific and early marker for sepsis and microbial infections. The immunoassay was performed on a bench-top system that fulfilled all the necessary characteristics of a POCT application, including a short measurement time (<9 min), no sample pre-treatment requirements and application directly near patients. New rat monoclonal antibodies targeting PCT were screened and characterized. The best combinations of antibodies were then integrated into single-use cartridges, and the reduction of nonspecific binding was achieved by supplying suitable additives. Moreover, human recombinant PCT (hrPCT) for use as a standard was developed in the native form of hPCT in plasma (PCT1-116, PCT3-116). The assay achieves the required sensitivity range in human plasma to allow reliable differentiation between healthy persons and varying stages of infection severity (LOD=0.04 ng/mL; LOQ=0.12 ng/mL). Furthermore, the developed PCT assay can be applied in whole human blood with an adequate sensitivity (LOD=0.02 ng/mL; LOQ=0.09 ng/mL). To the best of our knowledge, this is the first diagnostic test for sepsis to use whole blood, which is a crucial requirement for POCT. We were able to detect native PCT in patient samples and showed a good correlation (R(2)=0.988) with the results of the Kryptor(®) device from BRAHMS, a state of the art device for the detection of PCT.

Screening and characterization of new monoclonal anti-benzo[a]pyrene antibodies using automated flow-through microarray technology.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10 ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect on humans. In the present investigation, mice were immunized with B[a]P-bovine serum albumin conjugates and 110 generated hybridoma cell lines screened by different techniques to identify clones that produce anti-B[a]P antibodies. Subsequently, a new automated flow-through biochip noncompetitive direct chemiluminescence immunoassay (CLEIA) was compared with conventional indirect and direct enzyme-linked immunosorbent assays (ELISAs). It was demonstrated that the microchip-based screening method compared to ELISA was fast and very sensitive with use of only nanoliter volumes of supernatant. Forty clones could be evaluated in less than 5 min. Six high affinity monoclonal antibodies with different cross-reactivities (CR) for individual PAHs were identified by the chip-based assay and indirect microtiter plate ELISA. In comparison, the direct ELISA in the microtiter plate failed to identify three of these clones. The four antibodies with the highest affinity had half maximum inhibitory concentrations (IC(50) values) between 0.31 and 0.92 µg/L for B[a]P. Affinity constants of these four antibodies were determined by surface plasmon resonance using a water soluble B[a]P-peptide. The observed CR pattern of the four monoclonal antibodies for 16 tested PAHs was quite different. Only one specific antibody for B[a]P was observed, while others were more suitable for class-specific PAH determination.