Evaluation of PEG-L-asparaginase in asparagine suppression and anti-drug antibody development in healthy Beagle dogs: A multi-phase preclinical study.

L-asparaginase is a frequently used drug in the treatment of canine malignant lymphoma. Since production and availability of native E. coli-derived L-asparaginase are limited, PEG-L-asparaginase (PEG-ASP) is an alternative. However, recommended doses and dosing intervals are mainly empirically determined. A multi-phase clinical dose-finding study with seven healthy Beagle dogs was conducted to find the minimum effective dose and, potentially, a dosing interval for PEG-ASP in dogs. Plasma concentrations of amino acids and PEG-ASP activity were measured at various time points after administration of different doses of PEG-ASP. Anti-PEG and anti-asparaginase antibody titres were measured. Administration of 10 IU/kg PEG-ASP resulted in asparagine depletion in all dogs, albeit for various durations: for 9 days in all dogs, 15 days in five dogs, 21 days in three dogs and 29 days in one dog. Asparagine suppression occurred at PEG-ASP plasma concentrations < 25 IU/L. Subsequent administrations of a second and third dose of 20 IU/kg and 40 IU/kg PEG-ASP resulted in asparagine suppression at < 9 days in five dogs, accompanied by the development of antibodies against PEG and L-asparaginase. Two dogs with prolonged asparagine suppression after the second and third administration did not develop antibodies. Marked individual variation in the mechanism and duration of response to PEG-ASP was noted. Antibody formation against PEG-ASP was frequently observed and sometimes occurred after one injection. This study suggests that PEG-ASP doses as high as the currently used dose of 40 IU/kg might not be needed in treatment of canine malignant lymphoma.

Mitochondrial Neurogastrointestinal Encephalopathy: Clinical, Biochemical and Molecular Study in Three Egyptian Patients.

Background: Mitochondrial Neurogastrointestinal Encephalopathy syndrome is a rare autosomal recessive disorder. The disease is caused by mutations in the thymidine phosphorylase gene. This article reports the clinical, biochemical and molecular findings in three Egyptian patients with Mitochondrial Neurogastrointestinal Encephalopathy sundrome from two different pedigrees. Subjects and Methods: The three patients were subjected to thorough neurologic examination. Brain Magtnetic Resonance Imaging. Histochemical and biochemical assay of the mitochondrial respiratory chain complexes in muscle homogenate was performed (1/3). Thymidine Phosphorylase enzyme activity was performed in 2/3 patients and Thymidine Phosphorylase gene sequencing was done (2/3) to confirm the diagnosis. Results: All patients presented with symptoms of severe gastrointestinal dysmotility with progressive cachexia, neuropathy, sensory neural hearing loss, asymptomatic leukoencephalopathy. Histochemical analysis of themuscle biopsy revealed deficient cytochrome C oxidase and mitochrondrial respiratory chain enzyme assay revealed isolated complex 1 deficiency (1/3). Thymidine Phosphorylase enzyme activity revealed complete absence of enzyme activity in 2/3 patients. Direct sequencing of Thymidine Phosphorylase gene revealed c.3371 A>C homozygous mutation. Molecular screening of both families revealed heterozygous mutation in both parents and 4 siblings. Conclusions: Mitochondrial Neurogastrointestinal Encephalopathy syndrome is a rare mitochondrial disorder with an important diagnostic delay. In case of pathogenic mutations in Thymidine Phosphorylase gene in the family, carrier testing and prenatal diagmosis of at risk members is recommended for early detection. The possibility of new therapeutic options makes it necessary to diagnose the disease in an early state.

Sources of the adventitious microflora of a smear-ripened cheese.

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Phylogeny and identification of Enterococci by atpA gene sequence analysis.

The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.

Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching.

Six new Vibrio-like isolates originating from different species of bleached and healthy corals around Magnetic Island (Australia) were investigated using a polyphasic approach. Phylogenetic analyses based on 16S rRNA, recA and rpoA gene sequences split the isolates in two new groups. Strains LMG 22223(T), LMG 22224, LMG 22225, LMG 22226 and LMG 22227 were phylogenetic neighbours of Photobacterium leiognathi LMG 4228(T) (95.6 % 16S rRNA gene sequence similarity), whereas strain LMG 22228(T) was related to Enterovibrio norvegicus LMG 19839(T) (95.5 % 16S rRNA gene sequence similarity). The two new groups can be distinguished from closely related species on the basis of several phenotypic features, including fermentation of d-mannitol, melibiose and sucrose, and utilization of different compounds as carbon sources, arginine dihydrolase activity, nitrate reduction, resistance to the vibriostatic agent O/129 and the presence of fatty acids 15 : 0 iso and 17 : 0 iso. The names Photobacterium rosenbergii sp. nov. (type strain LMG 22223(T)=CBMAI 622(T)=CC1(T)) and Enterovibrio coralii sp. nov. (type strain LMG 22228(T)=CBMAI 623(T)=CC17(T)) are proposed to accommodate these new isolates. The G+C contents of the DNA of the two type strains are respectively 47.6 and 48.2 mol%.

Enterococcus saccharominimus sp. nov., from dairy products.

Four isolates, which were obtained from Belgian, Moroccan and Romanian dairy products, constituted a homogeneous but unidentified taxon after screening with whole-cell protein fingerprinting. Complete 16S rRNA gene sequence analysis classified representative strains in the genus Enterococcus. Highest sequence similarities of 98.6 and 98.0 % were obtained with the species Enterococcus sulfureus and Enterococcus saccharolyticus, respectively. Growth characteristics, biochemical features, tRNA intergenic length polymorphism analysis, DNA-DNA hybridization and DNA G+C contents of selected strains demonstrated that they represent a single, novel Enterococcus species. It differs phenotypically from other enterococci in characteristics commonly considered as typical of this genus: no growth in 6.5 % NaCl or 0.4 % sodium azide, and no acid production from a wide range of carbohydrates. The name Enterococcus saccharominimus sp. nov. is proposed for this novel species; the type strain (LMG 21727(T)=CCM 7220(T)) was isolated from contaminated pasteurized cow's milk.

Use of recA as an alternative phylogenetic marker in the family Vibrionaceae.

This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.

Vibrio fortis sp. nov. and Vibrio hepatarius sp. nov., isolated from aquatic animals and the marine environment.

In this study, the taxonomic positions of 19 Vibrio isolates disclosed in a previous study were evaluated. Phylogenetic analysis based on 16S rDNA sequences partitioned these isolates into groups that were closely related (98.8-99.1 % similarity) to Vibrio pelagius and Vibrio xuii, respectively. DNA-DNA hybridization experiments further showed that these groups had <70 % similarity to other Vibrio species. Two novel Vibrio species are proposed to accommodate these groups: Vibrio fortis sp. nov. (type strain, LMG 21557(T)=CAIM 629(T)) and Vibrio hepatarius sp. nov. (type strain, LMG 20362(T)=CAIM 693(T)). The DNA G+C content of both novel species is 45.6 mol%. Useful phenotypic features for discriminating V. fortis and V. hepatarius from other Vibrio species include production of indole and acetoin, utilization of cellobiose, fermentation of amygdalin, melibiose and mannitol, beta-galactosidase and tryptophan deaminase activities and fatty acid composition.

Vibrio neptunius sp. nov., Vibrio brasiliensis sp. nov. and Vibrio xuii sp. nov., isolated from the marine aquaculture environment (bivalves, fish, rotifers and shrimps).

The fluorescent amplified fragment length polymorphism (FAFLP) groups A5 (21 isolates), A8 (6 isolates) and A23 (3 isolates) distinguished in an earlier paper (Thompson et al., Syst Appl Microbiol 24, 520-538, 2001) were examined in more depth. These three groups were phylogenetically related to Vibrio tubiashii, but DNA-DNA hybridization experiments proved that the three AFLP groups are in fact novel species. Chemotaxonomic and phenotypic analyses further revealed several differences among the 30 isolates and known Vibrio species. It is proposed to accommodate these isolates in three novel species, namely Vibrio neptunius (type strain LMG 20536T; EMBL accession no. AJ316171; G +C content of the type strain 46.0 mol%), Vibrio brasiliensis (type strain LMG 20546T; EMBL accession no. AJ316172; G + C content of the type strain 45.9 mol%) and Vibrio xuii (type strain LMG 21346T; EMBL accession no. AJ316181; G +C content of the type strain 46.6 mol%). These species can be differentiated on the basis of phenotypic features, including fatty acid composition (particularly 14:0 iso, 14:0 iso 3-OH, 16:0 iso, 16:0, 17:0 and 17:1 omega8c), enzyme activities and utilization and fermentation of various carbon sources.

Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov.

Thirty-four Acetobacter strains, representing Acetobacter aceti, Acetobacter pasteurianus, Acetobacter pomorum, Acetobacter peroxydans, Acetobacter lovaniensis, Acetobacter estunensis, Acetobacter orleanensis, Acetobacter indonesiensis and Acetobacter tropicalis, were subjected to a polyphasic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis and phenotypic characterization. Two novel species are proposed, Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. The type strains of these species are respectively LMG 1625T (= DSM 14362T = NCIB 8894T = ATCC 23765T) and LMG 1746T (= DSM 14337T).

Lactobacillus durianis sp. nov., isolated from an acid-fermented condiment (tempoyak) in Malaysia.

Lactic acid bacteria (LAB) are the predominant micro-organisms in tempoyak, a Malaysian acid-fermented condiment. In a study on the diversity of LAB in this product, three isolates could not be identified using SDS-PAGE of whole-cell proteins or API 50 CH. The taxonomic position of the three isolates was clarified in the present study. 16S rDNA sequencing classified a representative strain in the genus Lactobacillus, clearly separated from all known species, and most closely related to the Lactobacillus reuteri phylogenetic group. DNA-DNA hybridization experiments and an extensive phenotypic description confirm that the strains represent a single and separate novel species among the obligately heterofermentative lactobacilli. The three isolates are distinguished at the intra-species level by plasmid profiling, pulsed-field gel electrophoresis of macro-restriction fragments and biochemical features. The name Lactobacillus durianis sp. nov. is proposed for the novel taxon and the type strain is LMG 19193T (= CCUG 45405T).

Burkholderia cepacia genomovar VI, a new member of the Burkholderia cepacia complex isolated from cystic fibrosis patients.

A polyphasic taxonomic study was performed on 23 strains isolated from cystic fibrosis (CF) patients in the USA. These strains were tentatively identified as Burkholderia cepacia, Burkholderia vietnamiensis and Burkholderia or Ralstonia sp. using biochemical tests and 16S rDNA-based PCR assays. Visual comparison of protein profiles indicated that they belonged to a single new group ('group 13'). The polyphasic taxonomic data showed that 18 of these strains represent a new member of the B. cepacia complex, referred to in this report as B. cepacia genomovar VI, whereas the other five strains belonged to Burkholderia multivorans. By means of biochemical tests, B. cepacia genomovar VI strains can be separated from B. cepacia genomovars I and III, Burkholderia stabilis, B. vietnamiensis and Burkholderia gladioli, but not from B. multivorans. Separation of B. cepacia genomovar VI and B. multivorans is possible using AFLP (amplified fragment length polymorphism) fingerprinting and DNA-DNA hybridizations. Retrospective analysis of epidemiological and genotypic data suggests that strains of B. cepacia genomovar VI have been involved in chronic colonization of CF patients and have been spread from person to person.

Genomic diversity amongst Vibrio isolates from different sources determined by fluorescent amplified fragment length polymorphism.

The genomic diversity among 506 strains of the family Vibrionaceae was analysed using Fluorescent Amplified Fragments Length Polymorphisms (FAFLP). Isolates were from different sources (e.g. fish, mollusc, shrimp, rotifers, artemia, and their culture water) in different countries, mainly from the aquacultural environment. Clustering of the FAFLP band patterns resulted in 69 clusters. A majority of the actually known species of the family Vibrionaceae formed separate clusters. Certain species e.g. V. alginolyticus, V. cholerae, V. cincinnatiensis, V. diabolicus, V. diazotrophicus, V. harveyi, V. logei, V. natriegens, V. nereis, V. splendidus and V. tubiashii were found to be ubiquitous, whereas V. halioticoli, V. ichthyoenteri, V. pectenicida and V. wodanis appear to be exclusively associated with a particular host or geographical region. Three main categories of isolates could be distinguished: (1) isolates with genomes related (i.e. with > or =45% FAFLP pattern similarity) to one of the known type strains; (2) isolates clustering (> or =45% pattern similarity) with more than one type strain; (3) isolates with genomes unrelated (<45% pattern similarity) to any of the type strains. The latter group consisted of 236 isolates distributed in 31 clusters indicating that many culturable taxa of the Vibrionaceae remain as yet to be described.

A polyphasic reassessment of the genus Paenibacillus, reclassification of Bacillus lautus (Nakamura 1984) as Paenibacillus lautus comb. nov. and of Bacillus peoriae (Montefusco et al. 1993) as Paenibacillus peoriae comb. nov., and emended descriptions of P. lautus and of P. peoriae.

Seventy-seven strains representing 10 species in the Paenibacillus polymyxa 16S rRNA group and 3 other species that exhibit phenetic relatedness to members of this group, Bacillus lautus, "Bacillus longisporus," and Bacillus peoriae, were characterized genotypically and phenotypically by performing an amplified ribosomal DNA restriction analysis, a randomly amplified polymorphic DNA analysis, a fatty acid methyl ester analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, pyrolysis mass spectrometry, and API and other routine phenotypic tests. These analyses revealed distinct clusters representing Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus azotofixans, Paenibacillus durum, Paenibacillus larvae subsp. larvae, Paenibacillus larvae subsp. pulvifaciens, B. lautus, Paenibacillus macerans, Paenibacillus macquariensis, B. peoriae, P. polymyxa, and Paenibacillus validus, which confirmed the distinctness of these species, but appreciable within-species heterogeneity was observed in P. alvei, B. lautus, P. macerans, P. polymyxa, and P. validus. The type strain of Paenibacillus pabuli did not cluster with other strains of this species, and in several analyses a relationship between strains of P. pabuli and "B. longisporus" was observed. As the analyses showed that B. lautus and B. peoriae are closely related to the genus Paenibacillus, these species are reclassified as members of this genus.

Reclassification of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura 1984) Ash et al. 1994, a later subjective synonym of Paenibacillus (formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a subspecies of P. larvae, with emended descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens.

A polyphasic taxonomic study of four strains of Paenibacillus larvae and four strains of Paenibacillus pulvifaciens (including duplicates of both type strains) supported the reclassification of both former Bacillus species into one species, P. larvae. Our conclusions were based on morphological and Analytab Products (API) tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, gas chromatography of methylated fatty acids, pyrolysis mass spectrometry, DNA-DNA binding, and the following genomic fingerprinting methods: amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and AFLP analysis. The last method is a novel high-resolution DNA fingerprinting technique based on the selective amplification of restriction fragments. Despite more than 90% DNA relatedness between the strains studied, SDS-PAGE of whole-cell proteins, biochemical tests, and DNA fingerprinting (AFLP) distinguished between the P. larvae and P. pulvifaciens strains at the subspecies level. Taking this evidence along with differences in pathogenicity, we propose to reclassify the honeybee pathogens P. larvae and P. pulvifaciens as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. An emended description of the species and descriptions of the subspecies are given. The type strains are P. larvae subsp. larvae ATCC 9545 (LMG 9820) and P. larvae subsp. pulvifaciens NRRL B-3685 (LMG 6911 and ATCC 13537).

Paenibacillus (formerly Bacillus) gordonae (Pichinoty et al. 1986) Ash et al. 1994 is a later subjective synonym of Paenibacillus (formerly Bacillus) validus (Nakamura 1984) Ash et al. 1994: emended description of P. validus.

A polyphasic study in which we performed an amplified ribosomal DNA restriction analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, a gas chromatographic analysis of methylated fatty acids, pyrolysis mass spectrometry, a random amplified polymorphic DNA analysis, a phenotypic analysis, and an analysis of the levels of DNA binding of Paenibacillus gordonae and Paenibacillus validus strains (including both type strains) showed that these organisms form a homogeneous group and that the names P. gordonae and P. validus are therefore synonyms. P. validus has nomenclatural priority, and an emended description of this species is given; the type strain is strain LMG 11161 (= ATCC 43897).

Characterization and identification of Vagococcus fluvialis strains isolated from domestic animals.

Strains of Vagococcus fluvialis, a species of Gram-positive catalase-negative cocci, related to the genera Enterococcus and Carnobacterium, were isolated from various lesions of pigs, from lesions and tonsils of cattle and cats and from tonsils of a horse. Most lesion strains were isolated in mixed culture from animals with disease conditions unrelated to coccal infection. Certain differences with the species description of Vagococcus fluvialis were found: only a proportion of the strains was motile; many strains gave positive reactions to Voges-Proskauer, alkaline phosphatase and leucine arylamidase tests or produced acid from galactose and D-tagatose. SDS-PAGE of whole-cell protein patterns, however, confirmed the phenotypic identification. Guidelines for identification of Vagococcus fluvialis are given and an emended description of the species is proposed.