Károly Márialigeti

Kocuria palustris sp. nov. and Kocuria rhizophila sp. nov., isolated from the rhizoplane of the narrow-leaved cattail (Typha angustifolia)

Two Gram-positive, aerobic spherical actinobacteria were isolated from the rhizoplane of narrow-leaved cattail (Typha angustifolia) collected from a floating mat in the Soroksár tributary of the Danube river, Hungary. Sequence comparisons of the 16S rDNA indicated these isolates to be phylogenetic neighbours of members of the genus Kocuria, family Micrococcaceae, in which they represent two novel lineages. The phylogenetic distinctness of the two organisms TA68T and TAGA27T was supported by DNA-DNA similarity values of less than 55% between each other and with the type strains of Kocuria rosea, Kocuria kristinae and Kocuria varians. Chemotaxonomic properties supported the placement of the two isolates in the genus Kocuria. The diagnostic diamino acid of the cell-wall peptidoglycan is lysine, the interpeptide bridge is composed of three alanine residues. Predominant menaquinone was MK-7(H2). The fatty acid pattern represents the straight-chain saturated iso-anteiso type. Main fatty acid was anteiso-C15:0. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol and an unknown component. The DNA base composition of strains TA68T and TAGA27T is 69.4 and 69.6 mol% G+C, respectively. Genotypic, morphological and physiological characteristics are used to describe two new species of Kocuria, for which we propose the names Kocuria palustris, type strain DSM 11925T and Kocuria rhizophila, type strain DSM 11926T.

Wohlfahrtiimonas chitiniclastica gen. nov., sp. nov., a new gammaproteobacterium isolated from Wohlfahrtia magnifica (Diptera: Sarcophagidae)

New Gammaproteobacteria were isolated from 3rd stage fly larvae of the parasitic fly Wohlfahrtia magnifica. Phylogenetic analysis of the new isolates showed that these bacteria belong to a distinct lineage close to Ignatzschineria larvae, which was originally isolated from the same species of fly. The low similarity values in 16S rRNA gene sequences (93.8-94.8 %), and differences in fatty acid profiles, RiboPrint patterns, MALDI-TOF mass spectra of cell extracts, and physiological and biochemical characteristics differentiate the isolates from the type strain of Ignatzschineria larvae (DSM 13226T), and indicate that our isolates represent a new genus within the Gammaproteobacteria. The major isoprenoid quinone of the strains is Q8, the major fatty acids are C18 : 1 and C14 : 0, and the predominant polar lipids are phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine. The G+C content of the DNA of the type strain is 44.3 mol%. The name Wohlfahrtiimonas chitiniclastica gen. nov., sp. nov., is proposed for this novel genus and species. The type strain is S5T (=DSM 18708T=CCM 7401T).

Polyphasic bacterial community analysis of an aerobic activated sludge removing phenols and thiocyanate from coke plant effluent

Biological purification processes are effective tools in the treatment of hazardous wastes such as toxic compounds produced in coal coking. In this study, the microbial community of a lab-scale activated sludge system treating coking effluent was assessed by cultivation-based (strain isolation and identification, biodegradation tests) and culture-independent techniques (sequence-aided T-RFLP, taxon-specific PCR). The results of the applied polyphasic approach showed a simple microbial community dominated by easily culturable heterotrophic bacteria. Comamonas badia was identified as the key microbe of the system, since it was the predominant member of the bacterial community, and its phenol degradation capacity was also proved. Metabolism of phenol, even at elevated concentrations (up to 1500mg/L), was also presented for many other dominant (Pseudomonas, Rhodanobacter, Oligella) and minor (Alcaligenes, Castellaniella, Microbacterium) groups, while some activated sludge bacteria (Sphingomonas, Rhodopseudomonas) did not tolerate it even in lower concentrations (250mg/L). In some cases, closely related strains showed different tolerance and degradation properties. Members of the genus Thiobacillus were detected in the activated sludge, and were supposedly responsible for the intensive thiocyanate biodegradation observed in the system. Additionally, some identified bacteria (e.g. C. badia and the Ottowia-related strains) might also have had a significant impact on the structure of the activated sludge due to their floc-forming abilities.

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.

Morphologic, Host Specificity, and Molecular Characterization of a Hungarian Cryptosporidium meleagridis Isolate

ABSTRACT This study was undertaken in order to characterizeCryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis andCryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location ofC. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridisisolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridissequence recently described for a North Carolina isolate. Thus,C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) andCryptosporidium felis. Because birds are susceptible toC. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.

Detection and characterisation of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes.

We investigated the prevalence of Giardia and Cryptosporidium species and analysed the genotypes in 36 samples collected from different water sources and various geographic areas in Hungary. Samples were collected from drinking water and sewage treatment plants and from the recreation area of Lake Balaton. The (oo)cysts were purified according to the US EPA 1623 method and they were detected by immunofluorescence test (IFT). Genomic DNA was extracted from all samples and then the GDH target gene for Giardia and the SSUrDNA for both Giardia and for Cryptosporidium species were amplified by PCR. 24 out of 36 samples (67%) were Giardia positive and 15 (42%) were Cryptosporidium positive by IFT. PCR confirmed that 13 out of 36 samples (36%) were Giardia positive and 10 (28%) contained Cryptosporidium. Twelve Giardia and two Cryptosporidium PCR products were successfully sequenced. In seven samples G. lamblia Assemblage A and in one sample Assemblage B and in four cases Assemblages A and B have been found. In one sample C. parvum and in the other separate sample C. meleagridis were detected. Sequence analysis revealed a new subtype of G. duodenalis complex, clustered close to the Assemblage A group. This study provides the first report on simultaneous detection and genotyping of G. duodenalis and Cryptosporidium species from water supplies in Hungary.

Schineria larvae gen. nov., sp. nov., isolated from the 1st and 2nd larval stages of Wohlfahrtia magnifica (Diptera: Sarcophagidae).

Four bacterial strains were isolated from the fly larvae of an obligate parasitic fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae). These isolates were characterized by a polyphasic approach and represent a new lineage of gamma-Proteobacteria as their closest relative is Xylella fastidiosa (87.1% 16S rDNA similarity). The four strains are identical at the 16S rDNA level, the level of similarity between them, based on DNA-DNA hybridization, is high (97.8-102.5%) and they are similar in their physiological and biochemical characteristics, although they differ in their utilization of different sole carbon sources. All produce chitinase. They are obligately aerobic: no growth is detected under anaerobic conditions, even in the presence of NO3- as terminal electron acceptor. Their predominant respiratory quinone is Q-8. The G+C content of their DNA is 42 mol%. Their cell membrane contains phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and two unknown polar lipids. Their main fatty acids are C18:1, C16:0 and C14:0. To accommodate these bacteria, a new genus, Schineria gen. nov., with the type species Schineria larvae sp. nov., is proposed.

Diversity of Alkaliphilic and Alkalitolerant Bacteria Cultivated from Decomposing Reed Rhizomes in a Hungarian Soda Lake

Bacterial communities associated with decomposing rhizomes of Phragmites australis were investigated in Lake Fertő (Neusiedlersee, Hungary). Alkaliphilic and alkalitolerant strains were isolated on cellulose-containing alkaline medium spread with dilutions of scrapings taken from the surface of the decaying plant material. Fifty-one strains were grouped by numerical analysis based on physiological tests and BIOLOG sole carbon source utilization data. The strains identified by 16S rDNA sequence comparisons included members of low G+C Gram positives (Marinibacillus marinus, Bacillus cereus, and Exiguobacterium aurantiacum), high G+C Gram positives (Nesterenkonia halobia and Dietzia natronolimnea), α-proteobacteria (Pannonibacter phragmitetus), and γ-proteobacteria (Pseudomonas pseudoalcaligenes and Halomonas venusta). Most of the strains were characterized by aerobic chemoorganotrophic respiratory metabolism and utilized several different carbon sources, although no direct cellulolytic activity was observed. Results of the pH and salt tolerance tests revealed optimuma in most cases at pH 11 and at the presence of 2.5–5% NaCl. These bacteria probably occupy niches in the aerobic, alkaline, water-influenced environments on the decomposing reed surfaces.

Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food, gut and excrement of saprophagous larvae ofPenthetria holosericea (Diptera: Bibionidae): a pilot study

Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare the bacterial communities of the food, the gut sections (ceca, anterior and posterior midgut, hindgut) and the excrement of the litter feeding bibionid larvae ofPenthetria holosericea. For universal eubacterial primers ARDRA patterns were complex with only minor differences among samples. Taxon specific primers were also applied to characterize the samples. Fragment composition was transformed to presence/absence binary data and further analyzed. Cluster analysis revealed that bacterial communities of gut highly resembled each other with the exception of the ceca. ARDRA patterns of consumed leaves clustered together with the intact leaves but differed from those of the excrement. ARDRA results were compared with microbial community structure based on phospholipid fatty acid (PLFA) fingerprints. The cluster analysis of PLFA (presence/absence binary) data resulted in a pattern similar to the ARDRA data. The PCA analysis of PLFA relative content separated microbial communities into five groups: (1) anterior and posterior midgut, (2) hindgut, (3) ceca, (4) consumed and intact litter, (5) excrement. Both methods indicated that conditions in the larval gut result in formation of a specific microbial community which differs from both the food and excrement ones. Particularly ceca — (blind appendages, harbor very specific microbial community) are divided from the rest of the gut by perithropic membrane.