Polyamine distribution profiles in the eighteen genera phylogenetically located within the Flavobacterium-Flexibacter-Cytophaga complex

Cellular polyamines of eighteen genera belonging to the Flavobacterium-Flexibacter-Cytophaga complex were analysed by ion exchange liquid chromatography. Homospermidine was the major polyamine in the genera Bergeyella, Riemerella, Ornithobacterium, Weeksella, Capnocytophaga, Polaribacter and Psychroflexus belonging to the family Flavobacteriaceae. In the family Spirosomaceae, Runella, Spirosoma and Flectobacillus species contained spermidine whereas Cyclobacterium species contained homospermidine. Within a divergent cluster, Haliscomenobacter and Lewinella species contained spermidine whereas Saprospira grandis contained agmatine alone. The major polyamine of Chitinophaga and Sporocytophaga species was homospermidine. Flexithrix dorotheae contained spermidine. Microscilla marina, the type species of the genus Microscilla, contained spermidine and cadaverine. However, 'Microscilla sericea' contained homospermidine, 'Microscilla furvescens' contained spermidine, and 'Microscilla arenaria' lacked all polyamines. Polyamine profiles serve as a phenotypic chemotaxonomic marker for the reclassification of the genera belonging to the complex.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and 1B comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and IB comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

In Vitro Susceptibility of Selected Bacteria to Cefaclor

Cefaclor is an orally absorbed cephalosporin antibiotic chemically and pharmacologically similar to cephalexin. It appears to be more active than cephalexin against susceptible strains. The in vitro sensitivity of 230 clinical bacterial isolates to cefaclor was studied. Most isolates of S. aureus, K. pneumoniae, E. coli, and indole negative Proteus species were inhibited at clinically attainable serum and urine concentrations. Like cephalexin, cefaclor was less active against isolates of Enterobacter species, indole positive Proteus species and enterococci although many of these isolates were inhibited at concentrations achievable in urine.     

Proposal to transfer 'Aegyptianella ranarum', an intracellular bacterium of frog red blood cells, to the family Flavobacteriaceae as 'Candidatus Hemobacterium ranarum' comb. nov

'Aegyptianella ranarum' (order Rickettsiales), an ultrastructurally defined small, Gram-negative rod, is known to replicate in the red blood cells of frogs. Heretofore, this bacterium has not been characterized genetically. We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of 'A. ranarum' from a Canadian frog blood specimen. In situ hybridization (with an 'A. ranarum' 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that 'A. ranarum' forms cytoplasmic inclusions in frog erythrocytes. blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both 'A. ranarum' genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum, a bacterium in the family Flavobacteriaceae. In contrast, 'A. ranarum' 16S rRNA shared only 61% identity with Aegyptianella pullorum. Phylogenetic analyses of these genes using phylip supported 'A. ranarum' as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa, rather than C. meningosepticum. We propose to classify 'Aegyptianella ranarum' as 'Candidatus Hemobacterium ranarum' in the family Flavobacteriaceae. Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.     

Isolation and characterization of anaerobic indole- and skatole-degrading bacteria from composting animal wastes

Four species of indole-degrading Clostridium and 3 species of skatole-degrading Clostridium were isolated from piggery or chicken manure composting processes. Since type strains of respective isolates did not degrade these compounds, the degradability of the compounds was a novel characteristic. All isolates were mesophilic. The maximum growth allowance concentrations of these isolates were 300 to 800 mg/l in indole and 100 to 300 mg/l in skatole. All isolates showed better growth and utilization of indolic compounds in nutrient-rich medium than in minimal medium. Skatole-degrading isolates degraded some substituted indoles tested, 3-indoleacetic acid, indole and oxindole, but did not degrade 1-methylindole, 2-methylindole, isatin or anthranilic acid. On the other hand, indole-degrading isolates degraded only oxindole. The growth of Clostridium malenominatum A-3 was inhibited by a low concentration (0.005%) of indole or skatole, even when 200-fold excess glucose was present in the medium. When 0.03% indole or skatole was added to the medium, C. malenominatum A-3 showed a lag phase for about 10 and 70 h, respectively. When 0.01% of these compounds was added to the medium, the uptake of glucose was inhibited. C. malenominatum A-3 degraded these compounds under nutrient-rich and minimal conditions.     

Production of indolic compounds by rumen bacteria isolated from grazing ruminants

AIM: To screen rumen bacterial cultures and fresh ruminal isolates for indole and skatole production. METHODS AND RESULTS: Culture collection strains and fresh bacterial isolates from rumen contents of sheep and dairy cows were screened for the production of indolic compounds. Clostridium aminophilum FT, Peptostreptococcus ssp. S1, Fusobacterium necrophorum D4 produced indole and Clostridium sticklandii SR produced indoleacetic acid. Fresh isolates from sheep (TrE9262 and TrE7262) and dairy cows (152R-1a, 152R-1b, 152R-3 and 152R-4) produced indole, indolepropionic acid, tryptophol and skatole from the fermentation of tryptophan and indoleacetic acid. Glucose altered the indolic compounds produced in some, but not all, isolates. TrE7262 and 152R-4 were identified as Clostridium sporogenes and 152R-1b as a new Cl. aminophilum strain. Isolates TrE9262, 152R-1a and 152R-3 were not closely related to any described species but belong to Megasphaera, Prevotella and Actinomyces genera, respectively. CONCLUSIONS: Rumen bacteria that produced a range of indolic compounds were identified. Some isolates are distinct from the previously described bacteria and may represent novel species. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations will contribute to understanding skatole and indole formation in the rumen and will lead to methods that control the formation of indolic compounds in pasture-grazed ruminants.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Characterization of Tannin-tolerant Bacterial Isolates from East African Ruminants

Several tannin-tolerant bacteria were isolated from enrichment cultures of rumen microflora of bush duiker, giraffe, Grant's gazelle, sheep, and goat, and established in medium containing crude tannin extracts or tannic acid. The isolates were characterized by classical and molecular methods. The isolates were also tested for the presence of tannin acylhydrolase. Characterization by restriction fragment length polymorphism of the 16S rRNA–PCR product was performed withAlu 1, Dde 1, Msp 1, and Taq 1. Amplified fragment length polymorphism analysis was performed only on the isolates that were curved rods. The nucleotide sequence of PCR products derived from the 16S rRNA genes of the isolates was determined. The classical characterization suggested that, with one exception all the curved rods isolates wereSelenomonas and the coccus was a Streptococcus. Only Selenomonas -like isolates had tannin acylhydrolase activities. One isolate lost the ability to completely hydrolyze tannins after prolonged storage at −70°C. The restriction fragment length polymorphism profiles suggested that the Selenomonas -like isolates exhibited heterogeneity in the ribosomal RNA locus. The coccus had the same profiles as Streptococcus caprinus, while the straight rods appeared to be similar to each other. Amplified fragment length polymorphism analysis suggested that the Selenomonas -like isolates clustered into two major groups. The 16S rRNA sequences of the coccus clustered with that ofStreptococcus species and the Selenomonas -like isolates exhibited a high level of similarity withSelenomonas ruminantium , while the straight rods clustered with Klebsiella species accessions in the databases. A partial 16S sequence strongly indicated that one of the isolates was Butyrivibrio fibrisolvens.     

Polymorphism of the major surface epitope of the CopB outer membrane protein of Moraxella catarrhalis

The CopB outer membrane protein has been considered a vaccine candidate for the prevention of infections due to Moraxella catarrhalis. Monoclonal antibody 10F3 recognizes whole cells of about 70% of clinical isolates, suggesting that this epitope is reasonably conserved. To determine whether CopB has other surface epitopes, we analyzed M. catarrhalis isolates using polyclonal sera against recombinant CopB proteins from a 10F3 positive isolate and a 10F3 negative isolate, and polyclonal sera against synthetic peptides that contained the sequence corresponding to the 10F3 epitope region of three different isolates. Extensive cross-reactivity was observed with the anti-CopB sera towards purified recombinant CopB proteins in Western blot and antigen ELISA, implying that antigenic regions common to both proteins were present. However, anti-CopB sera resembled anti-CopB peptide sera in exhibiting similar binding specificity to whole cells, segregating M. catarrhalis isolates into four CopB groups. We subsequently cloned and sequenced the copB genes from representative isolates. The deduced CopB amino acid sequences and the degree of sequence identity also demonstrated the existence of the same four CopB groups. Each of the four groups had a unique sequence in the 10F3 epitope region and a fifth group had the epitope deleted. The polymorphism of the major surface epitope prompts further consideration regarding the utility of CopB as a vaccine component as well as the design of an efficacious CopB-based vaccine to achieve broad protection against Moraxella infection.     

Spodiobacter cordis gen. nov. sp. nov., a member of the family Flavobacteriaceae isolated from patients with infective endocarditis

Two gram‐negative, catalase‐negative, oxidase‐positive strains (PAGU 1467^T and PAGU 1468) isolated from patients with infective endocarditis were investigated to determine their taxonomic status. 16S rRNA gene sequence analysis indicated that the two strains were members of the Bergeyella‐Chryseobacterium‐Riemerella branch of the family Flavobacteriaceae. Strains PAGU 1467^T and PAGU 1468 were highly related to each other (98.8% 16S rRNA gene sequence similarity). Phylogenetically closely‐related species to PAGU 1467^T comprised Bergeyella zoohelcum (95.0% 16S rRNA gene sequence similarity), Riemerella anatipestifer (94.3%) and Cloacibacterium normanense (94.3%). The major fatty acids of the two isolates were iso‐C_15:0, iso‐C_17:0 3‐OH and iso‐C_15:0 3‐OH. The presence of C_16:0 3‐OH and iso‐C_15:0 2‐OH allowed these isolates to be distinguished from B. zoohelcum. Menaquinone MK‐6 was the only respiratory quinone in these organisms; this is a consistent characteristic of the family Flavobacteriaceae. The guanine‐plus‐cytosine content of the genomic DNA was 42.0%, which is higher than that of other close phylogenetic relatives. On the basis of their phenotypic properties and genetic distinctiveness, isolates PAGU 1467^T and PAGU 1468 were classified within the novel genus Spodiobacter, as Spodiobacter cordis gen. nov., sp. nov., which is also the type species. The type strain of S. cordis is PAGU 1467^T ( = CCUG 65564^T = NBRC 109998^T).     

The influence of milk and milk components on the attachment of bacteria to farm dairy equipment surfaces

Glass, rubber and stainless steel surfaces were exposed to various types of bacteria in the presence of milk and a number of milk components under both static and agitated incubation conditions. Numbers of bacteria attaching were enumerated by epifluorescence microscopy. Results were affected by the different bacterial types, the nature of the attachment surface and the substances in which the bacteria were suspended with a Moraxella -like species, stainless steel and lactose and non-casein protein solutions respectively resulting in greatest numbers of cells attaching. Agitation had no marked influence on attachment.     

Detection of false-positives among total and fecal coliform counts by factorial analysis of correspondence

Application of an analysis of correspondence to the biochemical characteristics of total and fecal coliforms isolated in the Ivory Coast permitted us to separate two small clusters of isolates different from the main clusters, which included isolates from human and animal feces. The isolates grouped in the small clusters were from water samples. An analysis of the biochemical characteristics which permitted the segregation of the "water-specific" isolates from the main clusters indicates that water-specific total coliforms were citrate positive, indole negative, and amygdaline positive. Water-specific fecal coliforms were either citrate positive, indole negative, amygdaline positive, and inositol negative or indole negative, amygdaline positive, and inositol positive. Any isolates not fitting the above patterns could be considered of fecal origin. If this observation is confirmed under temperate climates and for a greater number of isolates, these simple tests could be used to confirm the fecal origin of coliforms.     

Detection of false-positives among total and fecal coliform counts by factorial analysis of correspondence.

Application of an analysis of correspondence to the biochemical characteristics of total and fecal coliforms isolated in the Ivory Coast permitted us to separate two small clusters of isolates different from the main clusters, which included isolates from human and animal feces. The isolates grouped in the small clusters were from water samples. An analysis of the biochemical characteristics which permitted the segregation of the "water-specific" isolates from the main clusters indicates that water-specific total coliforms were citrate positive, indole negative, and amygdaline positive. Water-specific fecal coliforms were either citrate positive, indole negative, amygdaline positive, and inositol negative or indole negative, amygdaline positive, and inositol positive. Any isolates not fitting the above patterns could be considered of fecal origin. If this observation is confirmed under temperate climates and for a greater number of isolates, these simple tests could be used to confirm the fecal origin of coliforms.     

Distribution of blaOXA genes among carbapenem-resistant Acinetobacter baumannii nosocomial strains in Poland

Acinetobacter baumannii is an important nosocomial pathogen occurring particularly in intensive care (ICU) as well as burn therapy units (BTU). A. baumannii strains have emerged as resistant to almost all antimicrobial agents, including carbapenems. b-lactamase-mediated resistance is the most common mechanism for carbapenem resistance in this species. Carbapenem-hydrolysing class D b-lactamases - OXA are widespread among A. baumannii strains. It is suggested that ISAba1 plays an important role in drug resistance. The aims of the study were detection of OXA encoding genes and presence of ISAba1. The study included the total of 104 isolates of carbapenem-resistant A. baumannii, obtained from patients hospitalized in ICU and BTU of Specialized Hospital in Krakow. Multiplex PCR was applied for detection of selected OXA carbapenemases encoding genes. PCR analysis showed the presence of bla OXA-51-like gene and ISAba1 in all isolates. 46 strains carried bla OXA-51-like and bla OXA-23-like genes while 48 bla OXA-51-like and bla OXA-40-like genes. 3 isolates carried: bla OXA-51-like , bla OXA-23-like and bla OXA-40-like genes. 7 strains encoded an OXA-51-like carbapenemase but were negative for enzymes belonging to the other families tested. Comparative analysis of ICU and BTU isolates revealed the dominance of: bla OXA-51-like and bla OXA-40-like among ICU while bla OXA-51-like and bla OXA-23-like in BTU.     

The role of bla(OXA-like carbapenemase) and their insertion sequences (ISS) in the induction of resistance against carbapenem antibiotics among Acinetobacter baumannii isolates in Tehran hospitals

This study aimed to evaluate the occurrence and dissemination of bla(OXA-like) carbapenemase genes and their insertion sequences among Acinetobacter baumannii isolates, taken from different hospitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs. A total number of 100 non duplicate Acinetobacter baumannii with different origins, were isolated from patients with proved nosocomial infections at eight university hospital in Tehran city. Antimicrobial susceptibility of these strains was done by E-test against 7 antimicrobial agents according to CLSI guideline. PCR of bla(OXA-51-like), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), IS(ABA-1), IS(1133) was carried out by specialized primers and then these strains were typed by REP-fingerprinting. Colistin, imipenem and meropenem were the most sensitive antibiotics against Acinetobacter baumannii isolates with 96%, 51% and 51% sensitivity respectively. All the isolates had a bla(OXA-51-like) intrinsic to these species. The rates of bla(OXA-23), 23 and 58-like were 38%, 32% and 1% respectively. Coexistence of bla(OXA-51/23/24-like) was observed among 16% of these isolates. All bla(OXA-23-like) carbapenemase genes had only one IS(ABA1). REP fingerprinting showed 5 genotypes among carbapenem resistant isolates, 16 of them being genotype A. This study emphasized on the major role of bla(OXA-like) carbapenemase, particularly bla(OXA-23-like) carbapenemase and their IS(ABA1), in the dissemination of carbapenem resistant Acinetobacter baumannii. This study confirmed a presumptive role of IS element neighboring the carbapenemase gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates for the first time in Iran.     

Multiple bacterial infection ofAlexandrium catenella (Dinophyceae)

Chilean clones of Alexandrium catenella are shown to be infectedwith bacteria. The endocyto-plasmic bacteria have been isolatedand grown in culture. Using biochemical characterization, theywere tentatively identified as Aeromonas salmonicida, Flavobacteriumbreve, Pseudomonas diminuta, Pasteurella haemolytica, Proteusvulgaris, Pseudomonas putida, Pseudomonas versicularis and Moraxellasp.-like. In several instances, the analysis showed that A.catenella clones are simultaneously infected by different speciesof bacteria. Intracellular bacterial localization was demonstratedby confocal and electron microscope observations. Viabilityof intracellular bacteria was confirmed by using a specificdye that becomes fluorescent when it is reduced by electronsgenerated by respiration of live organisms. Thus, the bacteriaare alive and appear to be dividing inside the cells. Antibiotictreatment of A. catenella did not generate bacteria-free cells,leading instead to the killing of the host cells. Of all bacterialstrains isolated, only Moraxella sp.-like and P. diminuta arecapable of producing small amounts of saxitoxin, as detectedby HPLC. Toxic Moraxella sp.-like bacterium was isolated onlyfrom one A. catenella clone and confirmed by western blot analysis,suggesting that bacterial infection might be clone specific.     

Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.