Tracking the sources of psychrotrophic bacteria contaminating chicken cuts during processing.

The major aim of the study was to establish the routes via which spoilage associated psychrotrophic bacteria contaminate poultry products at a large processing plant located in Belgium. Environmental samples were collected consisting of samples of air and swabs of food contact surfaces. Product samples were also collected consisting of modified atmosphere packaged (MAP) chicken wings and legs, which were analyzed microbiologically on the same day they were produced as well as after their sell-by date. Psychrotrophic bacteria from these samples were subsequently clustered and identified by means of MALDI-TOF MS and 16S rRNA gene sequencing. Carnobacterium maltaromaticum was determined to dominate the spoilage flora of both wings and legs. Other psychrotrophic bacteria able to grow on MRS which were identified on expired wings and legs included Carnobacterium divergens, Brocothrix thermosphacta, Lactobacillus curvatus, and Lactobacillus brevis. These were determined to arise from food contact surfaces such as cutting blades, leg hooks, Ertalon and polyurethane conveyor belts, working tables, and the hands of the operators. Importantly, it was determined that cleaning and disinfection was largely inadequate. Air was also determined to be an important vector of psychrotrophic bacteria in the processing environment, potentially contaminating the products directly or indirectly.

Determination of the microbiological contamination in minced pork by culture dependent and 16S amplicon sequencing analysis.

Routine evaluation of bacterial contamination in minced pork is still mainly performed by the enumeration of indicator bacteria, including total aerobic colony count and E. coli, using standardized isolation methods. However, the bacterial community structure as well as the effect of the storage time and temperature on the aerobic plate count are largely unknown for this matrix. The aim of the study was to characterize the microbial community in minced pork by 16S rRNA amplicon sequencing compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA gene sequencing. Analysis of 14 unrelated samples showed that total aerobic counts determined at 30 °C and 7 °C showed no significant difference, but the richness was higher on PCA at 30 °C for 7 samples, equal in 5, and higher at 7 °C for 2 samples. Members of the genus Pseudomonas, along with the genera Brochothrix and Carnobacterium were commonly identified among both the mesophilic and psychrotrophic population. Comparing to 16S rRNA amplicon sequencing, some contrasting data were obtained. Except for Brochothrix spp. and Pseudomonas spp., that were abundant and always detected, genera obtained with the two methods in the same sample were not always the same. Comparison of different sample preparation techniques and DNA extraction methods demonstrated also in this matrix that different results on the microbial composition and complexity are obtained. Present data illustrate that the combined isolation and identification of isolates using MALDI TOF MS and 16S gene sequencing and overall community profiling using 16S rRNA amplicon sequencing provides complementary results and yields important insights into the complex relationship between microorganisms in a food.

Detection, isolation and characterization of Fusobacterium gastrosuis sp. nov. colonizing the stomach of pigs.

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6-8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2h exposure to air. Phylogenetic analysis based on 16S rRNA and gyrase B genes demonstrated that the isolates showed high sequence similarity with Fusobacterium mortiferum, Fusobacterium ulcerans, Fusobacterium varium, Fusobacterium russii and Fusobacterium necrogenes, but formed a distinct lineage in the genus Fusobacterium. Comparative analysis of the genome of the type strain of this novel Fusobacterium sp. confirmed that it is different from other recognized Fusobacterium spp. DNA-DNA hybridization, fingerprinting and genomic %GC determination further supported the conclusion that the isolates belong to a new, distinct species. The isolates were also distinguishable from these and other Fusobacterium spp. by phenotypical characterization. The strains produced indole and exhibited proline arylamidase and glutamic acid decarboxylase activity. They did not hydrolyse esculin, did not exhibit pyroglutamic acid arylamidase, valine arylamidase, α-galactosidase, ß-galactosidase, ß-galactosidase-6-phosphate or α-glucosidase activity nor produced acid from cellobiose, glucose, lactose, mannitol, mannose, maltose, raffinose, saccharose, salicin or trehalose. The major fatty acids were C16:0 and C18:1ω9c. The name Fusobacterium gastrosuis sp. nov. is proposed for the novel isolates with the type strain CDW1(T) (=DSM 101753(T)=LMG 29236(T)). We also demonstrated that Clostridium rectum and mortiferum Fusobacterium represent the same species, with nomenclatural priority for the latter.

Combined approach to the identification of clinically infrequent non-tuberculous mycobacteria in Argentina.

SETTING: Over 150 potentially pathogenic non-tuberculous mycobacteria (NTM) species have been described, posing an onerous challenge for clinical laboratory diagnosis. OBJECTIVE: To evaluate different approaches for the identification of 40 clinically relevant NTM isolates whose species were not reliably identified using our routine diagnostic workflow comprising phenotypic tests and hsp65 polymerase chain reaction restriction analysis. DESIGN: We used 1) sequencing analysis of four conserved gene targets: 16S rRNA, rpoB, hsp65 and sodA; 2) two commercial reverse hybridisation assays; and 3) protein analysis using matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). RESULTS: Combined, but not individual, sequence analysis allowed reliable species identification for 30/40 (75%) isolates, including species previously unknown to be circulating in Argentina. Commercial kits outperformed our routine identification in only 5/35 isolates, and misclassified many more. MALDI-TOF MS accurately identified species in 22/36 (61%) isolates and did not misidentify any. CONCLUSIONS: Commercial kits did not resolve the problem of species of NTM isolates that elude identification. Combined DNA sequence analysis was the approach of choice. MALDI-TOF MS shows promise as a powerful, rapid and accessible tool for the rapid identification of clinically relevant NTM in the diagnostic laboratory, and its accuracy can be maximised by building up a customised NTM spectrum database.

Antimicrobial susceptibility of rapidly growing mycobacteria using the rapid colorimetric method.

Drug susceptibility testing (DST) of rapidly growing mycobacteria (RGM) are recommended for guiding the antimicrobial therapy. We have evaluated the use of resazurin in Mueller-Hinton medium (MHR) for MIC determination of RGM and compared the results with those obtained with the reference standard broth microdilution in Mueller-Hinton (MH) and with the resazurin microtiter assay (REMA) in 7H9 broth. The MIC of eight drugs: amikacin (AMI), cefoxitin (FOX), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), linezolid (LZD), moxifloxacin (MXF) and trimethoprim-sulfamethoxazole (TMP-SMX) were evaluated against 76 RGM (18 species) using three methods (MH, MHR, and REMA) in a 96-well plate format incubated at 37 °C over 3-5 days. Results obtained in the MH plates were interpreted by the appearance of turbidity at the bottom of the well before adding the resazurin. MHR and 7H9-REMA plates were read by visual observation for a change in color from blue to pink. The majority of results were obtained at day 5 for MH and 1 day after for MHR and 7H9-REMA. However, the preliminary experiment on time to positivity results using the reference strain showed that the resazurin can be added to the MH at day 2 to produce the results at day 3, but future studies with large sets of strains are required to confirm this suggestion. A high level of agreement (kappa 1.000-0.884) was obtained between the MH and the MHR. Comparison of results obtained with 7H9-REMA, on the other hand, revealed several discrepancies and a lower level of agreement (kappa 1.000-0.111). The majority of the strains were resistant to DOX and TMP-SMX, and the most active antimicrobials for RGM were AMI and FOX. In the present study, MHR represented an excellent alternative for MIC determination of RGM. The results could be read reliably, more easily, and more quickly than with the classical MH method.

Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam.

AIMS: To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria (LAB) from traditional fermented foods. METHODS AND RESULTS: A total of 119 strains of LAB from fermented meat (nem chua) were analysed with both (GTG)(5)-PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)(5)-PCR and in MALDI-TOF MS; five species grouped alike for (GTG)(5)-PCR and for MALDI-TOF MS; however, differences in minimal similarity between the delineated (GTG)(5)-PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)(5)-PCR profiles; two species, each represented by a single MALDI-TOF cluster, were subdivided in the corresponding (GTG)(5)-PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analysed using MALDI-TOF MS. PheS gene sequencing was used for validation. CONCLUSIONS: MALDI-TOF MS is a powerful, fast, reliable and cost-effective technique for the identification of LAB associated with the production of fermented foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Food LAB can be identified using MALDI-TOF MS, and its application could possibly be extended to other food matrices and/or other food-derived micro-organisms.

Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhea, drinking water and foods.

A collection of Aeromonas isolates obtained over a three-year period in the same geographic area (León, NW of Spain) was characterized by (GTG)5-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and gyrB gene sequence analysis. The isolates originated from human diarrheal stools (29 isolates), potable water (13 isolates), rabbit meat (13 isolates) and marine fish (5 isolates). The distribution of Aeromonas species varied with the strain source. Aeromonas caviae HG4 and Aeromonas media HG5 were predominant in clinical and water isolates, respectively, whereas motile Aeromonas salmonicida HG3 strains were most frequently found in fish and meat. Molecular typing revealed several genotypic relationships among specific isolate subsets: (i) two clones of A. media HG5 persisted in drinking water over the study period, (ii) different patients harbored identical or closely related clones during several months, and (iii) clonal relatedness was observed in two sets of water and human isolates. The first of these sets comprised nine water isolates and two human A. media HG5 isolates, whereas the other one included a water isolate and a human isolate of A. caviae HG4. The latter finding suggests that Aeromonas transmission in the studied region followed a waterborne route. Interestingly, the three human isolates closely related to water isolates were recovered in a period of four days in June 2006 from non-related patients without underlying medical conditions that tested negative for other enteric pathogens. The data imply the transmission through contaminated water of strains of the A. caviae group that can produce disease in humans.

Burkholderia nodosa sp. nov., isolated from root nodules of the woody Brazilian legumes Mimosa bimucronata and Mimosa scabrella.

Three strains, Br3437(T), Br3461 and Br3470, were isolated from nitrogen-fixing nodules on the roots of Mimosa scabrella (Br3437(T)) and Mimosa bimucronata (Br3461, Br3470), both of which are woody legumes native to Brazil. On the basis of 16S rRNA gene sequence similarities, all the strains were shown previously to belong to the genus Burkholderia. A polyphasic approach, including DNA-DNA hybridizations, PFGE of whole-genome DNA profiles, whole-cell protein analyses, fatty acid methyl ester analysis and extensive biochemical characterization, was used to clarify the taxonomic position of these strains further; the strains are here classified within a novel species, for which the name Burkholderia nodosa sp. nov. is proposed. The type strain, Br3437(T) (=LMG 23741(T)=BCRC 17575(T)), was isolated from nodules of M. scabrella.

Phenotypic and genotypic characterization of mutants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae.

A representative set of 19 mutants, with a known genealogy, of the virginiamycin producing strain Streptomyces virginiae 899 was investigated phenotypically and genotypically. Colour of the aerial and substrate mycelium were very variable both among spontaneous variants and those obtained after induced mutagenesis. At genotypic level, all mutants showed nearly identical BOX patterns, not reflecting the phenotypic heterogeneity observed. More than 40 years of forced mutational pressure did not cause huge chromosomal distortions but was most likely limited to base substitutions. The species S. virginiae, including besides producers of virginiamycin the type strain and non-type strains producing other bioactive compounds, is genomically heterogeneous on the basis of BOX-PCR fingerprinting and DNA-DNA hybridizations. The virginiamycin producing strain 899 does not belong to the species S. virginiae despite its phenotypic similarity to the latter.

Biodegradation of chemically modified flax fibers in soil and in vitro with selected bacteria.

The extent and rate of degradation of flax (Linum usitatissimum) fibers, both in the native state and after surface chemical modification (acetylation or poly(ethylene glycol), PEG, grafting), was investigated under laboratory conditions in two different biodegrading environments. Degradation of the fibers under aerobic conditions by the action of the microorganisms present in soil is assessed with the ASTM 5988-96 method by monitoring carbon dioxide evolution. In vitro biodegradation experiments were carried out by exposing the fibers to a pure culture of Cellvibrio fibrovorans bacteria and measuring the mass loss as a function of time. Despite the complexity of the system, the results of degradation in soil were satisfactorily reproducible, although the absolute rates were found to change in different experiments using the same soil. The degradation rate of acetylated fibers in soil nearly equals that of unmodified fibers, whereas in the pure culture, acetylated fibers biodegrade slower than native fibers. The opposite happens with the PEG-grafted fibers, which degrade slower than unmodified flax in soil and at a comparable rate upon in vitro exposure to the bacterial culture. The different biodegradation kinetics observed in the two biodegrading environments were attributed to differences of biocenoses, abiotic factors, and biodegradation assessing methods. Nevertheless, the final extent of biodegradation was the same for modified and unmodified fibers both in soil and in the pure culture, showing that the surface chemical modifications applied do not significantly affect biodegradability of the flax fibers.

Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis.

Vibrio sp. YB1T (=ATCC BAA-450T =LMG 20984T), the aetiological agent of tissue lysis of the coral Pocillopora damicornis, was characterized as a novel Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data (G + C content is 45.6 mol%), AFLP and GTG5-PCR genomic fingerprinting patterns and phenotypic properties, including the cellular fatty acid profile. The predominant fatty acids were 16:0 and 18:1 omega7c. The name Vibrio coralliilyticus sp. nov. is proposed for the novel coral-pathogenic species. In addition to strain YB1T, which was isolated from the Indian Ocean, five additional strains of V. coralliilyticus have been isolated, three from diseased P. damicornis in the Red Sea, one from diseased oyster larvae (Kent, UK) and one from bivalve larvae (Brazil). The six V. coralliilyticus strains showed high genotypic and phenotypic similarities and all were pathogenic to P. damicornis. The closest phylogenetic neighbours to V. coralliilyticus are Vibrio tubiashii, Vibrio nereis and Vibrio shilonii.

Bacterial Blight and Dieback of Eucalyptus Species, Hybrids, and Clones in South Africa.

During 1998, a new disease appeared on trees representing a Eucalyptus grandis × E. nitens (GN) hybrid in a nursery in KwaZulu/Natal. The disease has subsequently spread to other Eucalyptus species, hybrids, and clones in nurseries and plantations throughout South Africa. Typical symptoms of the disease include dieback of young shoots and leaf blight. This ultimately leads to stunting of trees. The objective of this study was to isolate and identify the causal agent of the disease. A bacterium was consistently isolated from infected tissue. Pathogenicity tests were undertaken with a range of bacterial strains. Four pathogenic strains were selected from different geographical regions and Eucalyptus hosts for further study. The bacterium causing Eucalyptus leaf and shoot blight is gram negative and rod-shaped, varying in size from 0.5 to 0.75 µm wide and 1.0 to 2.0 µm long. Colonies of this bacterium have a yellow pigment. The results from the Biolog tests identified the bacterium as Pantoea agglomerans with a similarity index of 0.315. The 16S rDNA sequences of the purported Pantoea sp. were compared with those of other related Enterobacteriaceae from GenBank/EMBL. Phylogenetic analysis using PAUP revealed that the isolates group together with P. agglomerans, P. ananatis, and P. stewartii subsp. stewartii. The fatty acid profiles and phenotypic characteristics of the new pathogen are similar to P. ananatis, and % G + C is within the range of this species. DNA:DNA hybridization between the four strains and the type strain of P. ananatis conclusively showed that the bacterium causing blight and dieback of Eucalyptus in South Africa belongs to this species. This is the first report in which P. ananatis has been found as a causal agent of a disease on Eucalyptus.

Natural cellulose fibers: heterogeneous acetylation kinetics and biodegradation behavior.

Steam-exploded fibers from flax (Linum usitatissimum) are heterogeneously acetylated using acetic anhydride and sulfuric acid as catalyst, with the aim to modify the surface properties without changing fiber structure and morphology. The acetylation reaction follows first-order kinetics up to a reaction time that depends on catalyst concentration (15 h when using 0.4 vol % of H(2)SO(4) or 50 h with 0.1 vol %). The fibers undergo no structural and/or morphological changes under either reaction condition. On the contrary, surface damage and structural modifications appear after longer reaction times, when the reaction kinetics change. The extent of biodegradation of acetylated fibers, evaluated from the weight percent remaining after 13 days of exposure to previously isolated cellulolytic bacteria Cellvibrio sp., decreases with increasing acetylation degree. After biodegradation the fibers show a higher acetyl content than before the experiment, indicating that the bacteria preferentially biodegrade unsubstituted cellulose, though also acetylated chains are cleaved. Biodegradable acetylated cellulose fibers with modified surface chemistry and unchanged structure are obtained for applications as polymer composite reinforcements.

Enforced expression of GATA-3 severely reduces human thymic cellularity.

Following bone marrow transplantation, patients often suffer from immune incompetence by reduced or late T cell development. Moreover, adult bone marrow stem cells have a lower capacity to generate T cells compared with fetal liver- and umbilical cord blood-derived progenitors. Therefore, enhancing thymic-dependent T cell generation might hold great therapeutic potential. GATA-3 is a transcription factor that is essential in T cell development. In this study we examined the therapeutic potential of GATA-3 to enhance T cell generation by overexpressing GATA-3 in T cell progenitors followed by fetal thymic organ culture (FTOC). We observed that early during FTOC, there was an enhanced differentiation toward the double positive stage of T cell development. From day 10 of FTOC, however, overexpression of GATA-3 induced a severe reduction in thymic cellularity, which probably correlates with the absence of a functional TCR-beta chain. We further show that the frequency of apoptosis was increased in GATA-3-transduced thymocytes. Despite the absence of a functional TCR-beta chain, GATA-3 transduced progenitors were able to differentiate into CD8beta(+) double positive thymocytes. This study shows that a strictly regulated expression of GATA-3 is essential for normal T cell development and this puts severe restrictions on the potential therapeutic use of continuously overexpressed GATA-3.

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor.

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.

Comparison of the antimicrobial tolerance of oxytetracycline-resistant heterotrophic bacteria isolated from hospital sewage and freshwater fishfarm water in Belgium.

The aim of this study was to investigate the relationship between antimicrobial tolerance and taxonomic diversity among the culturable oxytetracycline-resistant (Ot(r)) heterotrophic bacterial population in two Belgian aquatic sites receiving wastewater either from human medicine or from aquaculture. The study of Ot(r) heterotrophs and mesophilic Aeromonas spp. allowed comparison of tolerance data at the intergenus as well as at the intragenus level. In total, 354 independently obtained Ot(r) isolates were subjected to antimicrobial tolerance testing and identified by GLC analysis of their cellular fatty acid methyl esters (FAMEs), by API 20E profiling and/or by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) DNA fingerprinting. In general, Ot(r) hospital heterotrophs displayed a higher frequency (84%) of ampicillin (Amp) tolerance compared to the Ot(r) heterotrophs from the freshwater fishfarm site (22%). FAME results indicated that this effect was linked to the predominance of intrinsically ampicillin-resistant Ot(r) Aeromonas strains over representatives of Acinetobacter and Escherichia coli within the hospital strain set. Among the Ot(r) mesophilic Aeromonas strain set, the global tolerance profiles of the two sites only differed in a higher number of kanamycin (Kan) -tolerant strains (43%) for hospital aeromonads in comparison with the fishfarm aeromonads (8%). To some extent, this finding was correlated with the specific presence of Aeromonas caviae DNA hybridisation group (HG) 4. Collectively, these results suggest that the profiles for Amp and Kan tolerance observed in both sites arose from taxonomic differences in the culturable Ot(r) bacterial population at the generic or subgeneric level. In addition, our identification data also revealed that Enterobacter sp., Stenotrophomonas maltophilia, and A. veronii biovar sobria HG8 may be considered potential indicator organisms to assess microbial tolerance in various compartments of the aquatic environment.

Characterization of facultative oligotrophic bacteria from polar seas by analysis of their fatty acids and 16S rDNA sequences.

One hundred and seventy three bacterial strains, isolated previously after enrichment under oligotrophic, psychrophylic conditions from Arctic (98 strains) and Antarctic seawater (75 strains), were characterized by gas-liquid chromatographic analysis of their fatty acid compositions. By numerical analysis, 8 clusters, containing 2 to 59 strains, could be delineated, and 8 strains formed separate branches. Five clusters contained strains from both poles, two minor clusters were confined to Arctic isolates, and one cluster consisted of Antarctic isolates only. The 16S rRNA genes from 23 strains, representing the different fatty acid profile clusters and including the unclustered strains, were sequenced. The sequences grouped with the alpha and gamma Proteobacteria, the high percent G+C gram positives, and the Cytophaga-Flavobacterium-Bacteroides branch. The sequences of strains from 4 clusters and of 7 unclustered strains were closely related (sequence similarities above 97%) to reference sequences of Sulfitobacter mediterraneus, Halomonas variabilis, Alteromonas macleodii, Pseudoalteromonas species, Shewanella frigidimarina, and Rhodococcus fascians. Strains from the other four clusters and an unclustered strain showed sequence similarities below 97% with nearest named neighbours, including Rhizobium, Glaciecola, Pseudomonas, Alteromonas macleodii and Cytophaga marinoflava, indicating that the clusters which they represent form as yet unnamed taxa.

Isolation and identification of cellulolytic bacteria involved in the degradation of natural cellulosic fibres.

In search for bacterial cultures that are able to rapidly degrade cellulosic plant fibres in vitro, 77 cellulolytic strains were isolated from Belgian and Czech soils after enrichment on flax or sisal fibres as sole sources of carbon. The strains were characterized using fatty acid analysis, and 74 strains were grouped into three major clusters by numerical analysis. The first major cluster contained Cellulomonas strains. Within this cluster three subclusters could be delineated by principal component analysis, that were recognized by their fatty acid compositions as Cellulomonas gelida, Cellulomonas biazotea and Cellulomonas cellulans, containing 9, 8 and 13 strains respectively. The second major cluster, with 9 strains, was assigned to Flavobacterium johnsoniae. The 34 strains of the third cluster could not be identified by commercial identification systems on the basis of their fatty acid profiles and API 20NE profiles. On the basis of their phenotypic characteristics they met the description of the genus Cellvibrio, their fatty acid profiles were similar to those of four authentic Cellvibrio mixtus strains, and the 16S rRNA genes from four representatives showed up to 97.8% sequence similarity to 16S rDNA from Cellvibrio mixtus ACM 2603. Three non-clustered strains were assigned to Curtobacterium flaccumfaciens, Achromobacter piechaudii and Pseudomonas mendocina. Two strains assigned to Cellvibrio were able to degrade several flax, broom and cotton fibres very rapidly in a standardized in vitro test, causing mass losses of 40 to 86% within 13 days of incubation, but not jute.

Reclassification of non-pigmented Erwinia herbicola strains from trees as Erwinia billingiae sp. nov.

Twenty-two Erwinia-like strains, isolated from trees since the late fifties and belonging to a distinct phenotypic group with resemblance to Pantoea agglomerans, were further characterized by conventional biochemical tests, the BIOLOG metabolic fingerprinting system and fatty acid analysis. Their phylogenetic positions were determined by comparing the 16S rRNA gene sequence of a representative strain to available sequences of Erwinia, Pantoea, Pectobacterium and Brenneria species. The strains were shown to belong to the genus Erwinia, with Erwinia rhapontici and Erwinia persicina as the closest phylogenetic relatives. The name Erwinia billingiae sp. nov. is proposed (type strain LMG 2613T) and a description of the species is given.