Brevundimonas balnearis sp. nov., isolated from the well water of a thermal bath.

A novel alphaproteobacterium was isolated from the well water of a thermal bath at Budapest, Hungary. Phylogenetic analysis of the novel strain showed that this bacterium belongs to a distinct lineage among the genus Brevundimonas. Based on the 16S rRNA gene sequence strain FDRGB2bT showed the highest sequence similarity values to Brevundimonas naejangsanensis BIO-TAS2-2T (97.35 %), Brevundimonas viscosa F3T (97.28 %), Brevundimonas vesicularis LMG 2350T (97.27 %), Brevundimonas nasdae GTC 1043T (97.14 %), Brevundimonas vancanneytii LMG 2337T (97.13 %) and Brevundimonas aurantiaca DSM 4731T (97.13 %). The newly isolated bacterium was strictly aerobic, and its optimum growth occurred at 20-30 °C, between pH 8-9 and without NaCl. Movement was with a single polar flagellum, but the cells could also produce stalks. The major isoprenoid quinone of strain FDRGB2bT was Q-10, the major cellular fatty acids were C18 : 1ω7c and C16 : 0, and the polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol, two unknown phospholipids and four unknown glycolipids. The characteristic diamino acid in its cell wall is meso-diaminopimelic acid. The G+C content of DNA of the type strain was 69.8 mol%. Strain FDRGB2bT (=DSM 29841T=NCAIM B.02621T) is proposed as the type strain of a novel species with the proposed name Brevundimonas balnearis sp. nov.

Sequence analysis of 16S rRNA, gyrB and catA genes and DNA-DNA hybridization reveal that Rhodococcus jialingiae is a later synonym of Rhodococcus qingshengii.

The results of 16S rRNA, gyrB and catA gene sequence comparisons and reasserted DNA-DNA hybridization unambiguously proved that Rhodococcus jialingiae Wang et al. 2010 and Rhodococcus qingshengii Xu et al. 2007 represent a single species. On the basis of priority R. jialingiae must be considered a later synonym of R. qingshengii.

Phreatobacter oligotrophus gen. nov., sp. nov., an alphaproteobacterium isolated from ultrapure water of the water purification system of a power plant.

Strains of a novel alphaproteobacterium were isolated from ultrapure water of a Hungarian power plant on a newly developed medium. Phylogenetic analysis of the 16S rRNA gene sequences of the novel strains showed that these bacteria belong to a distinct lineage far from any known taxa. Based on the 16S rRNA gene sequences, strains PI_31, PI_25 and PI_21(T) exhibited the highest sequence similarity to Bosea minatitlanensis AMX51(T) (93.43 %) and Bosea thiooxidans DSM 9653(T) (93.36 %); similarity to all other taxa was less than 93.23 %. Fatty acid profiles, matrix-assisted laser-desorption/ionization time-of-flight mass spectra of cell extracts as well as physiological and biochemical characteristics indicated that our strains represent a novel genus and species within the class Alphaproteobacteria. The major isoprenoid quinone of the strains was Q-10, the major cellular fatty acids were C18 : 1ω7c and 11-methyl C18 : 1ω7c and the polar lipid profiles of the strains contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and several unknown phospholipids and other lipids. The characteristic diamino acid in their cell wall was meso-diaminopimelic acid. The G+C content of DNA of the proposed type strain PI_21(T) was 68.9 mol%. A new genus and species, Phreatobacter oligotrophus gen. nov., sp. nov., is proposed to accommodate the strains. Strain PI_21(T) ( = DSM 25521(T) = NCAIM B 02510(T)) is the type strain of Phreatobacter oligotrophus.

Aquipuribacter hungaricus gen. nov., sp. nov., an actinobacterium isolated from the ultrapure water system of a power plant.

A Gram-positive actinobacterium, strain IV-75(T), was isolated by using R2A agar from the ultrapure water system of a power plant in Hungary. The strain exhibited a rod-coccus cell cycle, and was strictly aerobic, non-motile, catalase-positive and oxidase-negative. 16S rRNA gene sequence analysis revealed that strain IV-75(T) belonged to the suborder Micrococcineae and clustered with members of the family Intrasporangiaceae. Its closest phylogenetic neighbour was Arsenicicoccus bolidensis CCUG 47306(T) (94.3% 16S rRNA gene sequence similarity). The peptidoglycan of strain IV-75(T) contained meso-diaminopimelic acid and MK-10(H(4)) was the major menaquinone. The polar lipid pattern contained phosphatidylglycerol, two unidentified phospholipids, one glycolipid and several other lipid components. The major fatty acids were anteiso-C(15:0), C(18:1)ω9c and C(16:0). Based on the moderate levels of 16S rRNA gene sequence similarity to all members of the family Intrasporangiaceae and the unique combination of chemotaxonomic characteristics, strain IV-75(T) is considered to represent a novel species of a new genus, for which the name Aquipuribacter hungaricus gen. nov., sp. nov. is proposed. The type strain of Aquipuribacter hungaricus is IV-75(T) (=DSM 21674(T)=NCAIM B 02333(T)).

Applicability of the functional gene catechol 1,2-dioxygenase as a biomarker in the detection of BTEX-degrading Rhodococcus species.

AIMS: Catechol 1,2-dioxygenase is a key enzyme in the degradation of monoaromatic pollutants. The detection of this gene is in focus today but recently designed degenerate primers are not always suitable. Rhodococcus species are important members of the bacterial community involved in the degradation of aromatic contaminants and their specific detection could help assess functions and activities in the contaminated environments. To reach this aim, specific PCR primer sets were designed for the detection of Rhodococcus related catechol 1,2-dioxygenase genes. METHODS AND RESULTS: Primers were tested with genetically well-characterized strains isolated in this study and community DNA samples were used as template for Rhodococcus specific PCR as well. The sequences of the catabolic gene in question were subjected to multiple alignment and a phylogenetic tree was created and compared to a 16S rRNA gene based Rhodococcus tree. A strong coherence was observed between the phylogenetic trees. CONCLUSIONS: The results strongly support the opinion that there was no recent lateral gene transfer among Rhodococcus species in the case of catechol 1,2-dioxygenase. SIGNIFICANCE AND IMPACT OF THE STUDY: In gasoline contaminated environments, aromatic hydrocarbon degrading Rhodococcus populations can be identified based upon the detection and sequence analysis of catechol 1,2-dioxygenase gene.

First investigations into the prevalence of Cryptosporidium and Giardia spp. in Hungarian drinking water.

Safe drinking water is a top priority in preventing disease outbreaks and is of general concern to everyone. This study examines the occurrence of Cryptosporidium and Giardia in Hungarian drinking water supplies for the first time. A total of 76 raw and drinking water samples were examined using the U.S. EPA Method 1623. From these 15 of 34 (48.4%) raw water samples tested positive for Giardia and 7 (26.6%) for Cryptosporidium. Twelve of 45 (26.7%) drinking water samples were positive for Giardia and 6 (13.3%) for Cryptosporidium. Overall, Giardia cysts and/or Cryptosporidium oocysts were detected in 48% of the raw water samples and 35% of the drinking water samples. The highest levels in drinking water were found to be 3 oocysts/100 litres of Cryptosporidium and 63.6 cysts/100 litres for Giardia, enough to cause giardiasis. The highest levels in raw water were 1,030 cysts/100 litres for Giardia and 50 oocysts/100 litres for Cryptosporidium and higher oocyst densities were associated with source water receiving effluents from sewage treatment plants or originating from a forest environment. In addition to this monitoring, riverbank filtrated water and raw water from the River Danube in Budapest were monitored in order to ascertain protozoan removal efficiency of riverbank filtration (RBF). A total of 157 samples, including 87 samples from the River Danube and 70 samples post RBF, were examined. Cryptosporidium and Giardia were detected regularly in the river water but never in riverbank filtered water suggesting the effectiveness of RBF as a purification method. The occurrence of Cryptosporidium oocysts and Giardia cysts in the investigated water supplies may require the water utilities and water authorities in Hungary to apply additional monitoring and treatment and/or watershed controls.

Temporal distribution of Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary.

A survey was carried out over a 4-year period to describe the temporal distribution of three 'anthropophilic' tick species, Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary. Altogether 4658 adult ticks belonging to the three species were collected from 1931 red foxes (Vulpes vulpes) killed in an area of about 70,000 km(2) representing all major climatic areas of the country. The seasonal activity of the three species was different. I. ricinus ticks were most active between April and June with an activity peak in May. A less marked increase of activity was also observed in September and October. The highest activity of D. reticulatus ticks was seen between September and November with an activity peak in October, nevertheless, a marked increase of activity could also be observed in April. Small number of I. ricinus and D. reticulatus were collected in all other months. H. concinna ticks were active from May to July with an activity peak in June and completely disappeared between October and March. The temporal distribution of the three tick species might be used for predictions on the seasonality of tick-borne diseases in Hungary.

Evaluation of efficacy of entomopathogenic nematodes against larvae of Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae).

The blowfly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) is the primary agent of cutaneous myiasis of sheep in northern Europe, southern Africa, Australia and New Zealand. As the application of chemicals has several disadvantages, alternative control measures of traumatic myiasis of livestock must be developed. In this study, the use of entomopathogenic nematodes (EPNs) as potential biocontrol agents against second instar larvae of Lucilia sericata was considered. The following nematode species were tested: Heterorhabditis bacteriophora (IS 5, HHU 1, Hmol, HNC 1, HAZ 36, Hbrecon, HHU 2, HAZ 29, HHP 88, HHU 3, HHU 4 and HGua), Steinernema intermedia, NC513 strain of S. glaserii, S. anomali, S. riobrave, Steinernema sp. and 5 strains of S. feltiae (22, Vija Norway, HU 1, scp, and IS 6). None of the examined EPN species or strains showed larvicidal efficacy at 37 degrees C (no killing effect was observed in the case of the two heat-tolerant strains--H. bacteriophora and S.feltiae) against L. sericata larvae. At lower temperatures (20 degrees C and 25 degrees C) only strains of S. feltiae were found to be active. The overall odds ratios calculated for L. sericata maggots to contract S. feltiae nematode infection show significant (p < 0.05) effect only in the case of strains HU 1, 22 and IS 6. In the case of strains HU 1 and 22 parasitic forms of S. feltiae could be detected in the dead larvae of L. sericata. Strain IS 6 (and also Vija Norway at 20 degrees C) penetrated and killed fly larvae, but only adult forms of the nematode occurred in the cadavers.

Electron microscopic and molecular identification of Wolbachia endosymbionts from Onchocerca lupi: implications for therapy.

It was recently demonstrated that Wolbachia intracellular bacteria (alpha 2 proteobacteria, Rickettsiales) living in filarial nematodes are obligatory symbionts of their hosts. Herein, we report the electron microscopic and 16S ribosomal DNA-based (16S rDNA) identification of the endobacteria harboring in Onchocerca lupi. The worm nodules containing the nematodes were removed from three Hungarian dogs naturally infected with O. lupi. Wolbachia-like endobacteria were detected by electron microscopy in the lateral chords of both adult worms and microfilariae. The endosymbionts in O. lupi resemble in location, size, and morphology the wolbachiae found in other filariae. The presence of wolbachiae in O. lupi was also confirmed by PCR amplification of the 16S rDNA of the bacteria. The 16S rDNA-based phylogenetic analysis revealed that the endosymbionts of O. lupi infecting dogs belong to the supergroup C of Wolbachia pipientis and are not identical with those of other Onchocerca spp. sequenced so far. Since intermittent treatment with oxytetracycline has adulticid and microfilaricid activity by depletion of Wolbachia endobacteria, this antibiotic treatment regimen may offer an alternative of ivermectin or diethylcarbamazine in the suppression of postoperative microfilaridermia in Onchocerca-infected dogs and may prevent relapse.

Molecular phylogenetic analysis of Onchocerca lupi and its Wolbachia endosymbiont.

The morphology of Onchocerca lupi, responsible for canine ocular onchocercosis, is unique within the genus. Earlier analyses of the 5S ribosomal RNA gene spacer region sequence of the parasite and the 16S ribosomal RNA gene sequence of its Wolbachia endosymbiotic bacteria (Rickettsiales) supported the morphological and biological arguments that O. lupi is a distinct species. However, the exact phylogenetic position of O. lupi and its endosymbiont could not be unambiguously determined. Herein we report analyses based on the mitochondrial cytochrome oxidase I (COI) gene of the filarial species and the Wolbachia surface protein (wsp) and the bacterial cell-cycle ftsZ genes of their wolbachiae. Our results indicate that O. lupi separated from other Onchocerca spp. early in evolution. This is in line with the previous morphological analysis demonstrating that O. lupi is an atypical Onchocerca species showing both primitive and evolved characters. The phylogenetic trees generated for the COI sequences of filariae and the wsp and ftsZ sequences of their wolbachiae were congruent with each other, which supports the hypothesis that nematodes and their Wolbachia endobacteria share a long co-evolutionary history.

Morphologic and genetic characterization of Onchocerca lupi infecting dogs.

In the past decades, sporadic cases of ocular Onchocerca infection have been reported in canids in US and Europe. The present study was undertaken to provide a detailed description of the morphologic characteristics of adults and microfilariae and to characterize the 5S ribosomal rRNA gene (5S rDNA) spacer sequences of Onchocerca lupi causing canine onchocercosis. The morphology of O. lupi is unique within the genus, and morphology based cluster analysis indicates that O. lupi is not closely related to the members of domestic cattle or horse clades occurring in North America and Europe. Similarly, the signature of the 5S rDNA spacer sequences of O. lupi does not resemble any other Onchocerca 5S rDNA spacer sequences including those of the members of domestic cattle or horse clades. The adult and microfilarial morphology and sequence signature supports the biological arguments that a distinct species, O. lupi and not O. lienalis, is responsible for canine ocular onchocercosis.

Morphologic, host specificity, and genetic characterization of a European Cryptosporidium andersoni isolate.

This study was undertaken in order to characterize a Cryptosporidium muris-like parasite isolated from cattle in Hungary and to compare this strain with other Cryptosporidium species. To date, the large-type oocysts isolated from cattle were considered as C. muris described from several mammals. The size, form, and structure of the oocysts of the Hungarian strain were identical with those described by others from cattle. An apparent difference between the morphometric data of C. muris-like parasites isolated from cattle or other mammals was noted, which is similar in magnitude to the differences between Cryptosporidium meleagridis and Cryptosporidium felis or between Cryptosporidium serpentis and Cryptosporidium baileyi. The cross-transmission experiments confirmed the findings of others, as C. muris-like oocysts isolated from cattle fail to infect other mammals. The sequence of the variable region of small subunit (SSU) rRNA gene of the strain was 100% identical with that of the U.S. Cryptosporidium andersoni and C. andersoni-like isolates from cattle. The difference between the SSU rRNA sequence of bovine strains and C. muris is similar in magnitude to the differences between C. meleagridis and Cryptosporidium parvum anthroponotic genotype or between Cryptosporidium wrairi and C. parvum zoonotic genotype. Our findings confirm that the Cryptosporidium species responsible for abomasal cryptosporidiosis and economic losses in the cattle industry should be considered a distinct species, C. andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000.

Polyphasic typing of Cryptosporidium baileyi: a suggested model for characterization of cryptosporidia.

The present study was undertaken to characterize the oocyst morphology, host specificity, organ location, virulence, and sequences of the small subunit ribosomal RNA, 70-kDa heat shock protein, and oocyst wall protein genes of Cryptosporidium baileyi, and to compare this strain with other Cryptosporidium species. This study also aims to serve as a model for polyphasic (phenetic and genetic) characterization of Cryptosporidium species and strains. On the basis of these results, further genetic and phenetic characterization of an avian isolate is needed if the difference between the length or width, or both, of oocysts of an isolate and of C. baileyi is > or = 10% or if the difference between the oocyst shape index of the isolate and of C. baileyi is > or = 3% (or both). The isolate is infectious for mammals or lower vertebrates, or the host range is narrow, i.e., infectious only for some bird species; after oral or intratracheal inoculation, the parasites are not located in the cloaca and in the bursa of Fabricius or the respiratory tract; clinical disease or weight gain reduction can be observed after oral inoculation; the genetic distance for the examined gene between C. baileyi and the isolate is similar in magnitude to that observed between most closely related Cryptosporidium species.

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food, gut and excrement of saprophagous larvae of Penthetria 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 of Penthetria 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.

Bacteriological investigations on wound myiasis of sheep caused by Wohlfahrtia magnifica (Diptera: Sarcophagidae).

The aim of the present investigation was to get further information about obligate aerobic and facultatively anaerobic bacterial communities of the intact and Wohlfahrtia magnifica infested vulval region of sheep. The numbers of aerobic and facultatively anaerobic microorganisms were lower in samples taken from uninfested mucous membrane and myiatic wounds as well as in the wound fluid as compared to samples originating from the uninfested skin surface. Gram-negative bacteria belonging to the family Enterobacteriaceae were isolated only from the skin and mucous membrane of uninfested sheep. Gram-positive microorganisms dominated in all samples. The ratio of facultatively anaerobic bacteria was higher than 80% in the sample taken from a lesion containing third instar Wohlfahrtia larvae and in the wound discharge collected from a vulval wound free of maggots. It is suggested that there is a shift in the composition of the bacterial communities of vulva as staphylococci disappear from the wounds due to the presence of Wohlfahrtia larvae.

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya.

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.

Detection of potentially pathogenic bacteria in the drinking water distribution system of a hospital in Hungary.

The drinking water distribution system of a hospital was investigated using standard cultivation techniques, taxon-specific PCRs targeting pathogenic bacteria, denaturing gradient gel electrophoresis, cloning and sequencing. The results obtained verify the higher sensitivity of PCR compared to cultivation for detecting Legionella and Pseudomonas aeruginosa. Moreover, several other opportunistic pathogenic bacteria, such as Escherichia albertii, Acinetobacter lwoffi and Corynebacterium tuberculostrearicum, were detected, emphasizing that drinking water systems, especially those with stagnant water sections, could be the source of nosocomial infections.

Investigation of mineral water springs of Miercurea Ciuc (Csíkszereda) region (Romania) with cultivation-dependent microbiological methods.

Water samples of ten mineral water springs at Miercurea Ciuc (Csíkszereda) region (Romania) were examined during 2005-2006 using cultivation-dependent microbiological methods. The results of standard hygienic bacteriological tests showed that the Hargita Spring had perfect and five other springs had microbiologically acceptable water quality (Zsögöd-, Nagy-borvíz-, Taploca-, Szentegyháza- and Lobogó springs). The water of Borsáros Spring was exceptionable (high germ count, presence of Enterococcus spp.).Both standard bacteriological and molecular microbiological methods indicated that the microbiological water quality of the Szeltersz-, Nádasszék- and Délo springs was not acceptable. Bad water quality resulted from inadequate spring catchment and hygiene (low yield, lack of runoff, negligent usage of the springs, horse manure around the spring).The 16S rRNA gene-based identification of strains isolated on standard meat-peptone medium resulted in the detection of typical aquatic organisms such as Shewanella baltica, Aeromonas spp., Pseudomonas veronii, Psychrobacter sp,. Acinetobacter spp. and allochthonous microbes, like Nocardia, Streptomyces, Bacillus, Microbacterium , and Arthrobacter strains indicating the impact of soil. Other allochthonous microbes, such as Staphylococcus spp., Micrococcus sp., Lactococcus sp., Clostridium butyricum, Yersinia spp., Aerococcus sp., may have originated from animal/human sources.

Diversity and activity of cultivable aerobic planktonic bacteria of a saline Lake located in Sovata, Romania.

Aerobic bacterial strains from the salt water of Lake Red (Sovata, Romania) were cultivated. More than half of the 80 strains were G(-) and formed motile straight rods. Only a few strains produced acid from D-glucose and reduced nitrate to nitrite. Optimum NaCl concentration for growth varied between 5 and 15 % in the majority of the strains, so the isolates were regarded moderately halophilic. On the basis of the 16S rRNA gene sequence similarity almost half of the strains were identified as members of genus Halomonas. Other strains belonged to genera Marinobacter, Psychrobacter, Serratia, Morganella (γ-Proteobacteria), Bacillus, Exiguobacterium, Planococcus (Firmicutes), and Arthrobacter, Micrococcus, Microbacterium, and Nesterenkonia (Actinobacteria).

Molecular biological investigations on the bacterial communities of curative well waters of Harkány Spa.

Bacterial communities from the sulfide containing curative well waters of Harkány Spa (Hungary) were investigated by cultivation independent molecular cloning and Denaturing Gradient Gel Electrophoresis (DGGE) methods between 2006 and 2008. The DGGE profiles of the bacterial communities originated from the wells of lukewarm waters showed seasonal similarities and were highly different from the thermal well. From the four clone libraries 22 different eubacterial species or genera were identified by sequence analysis. The majority of the clones of the lukewarm waters belonged to unidentified Epsilon-proteobacteria, Desulfocapsa sp. and Thiothrix spp., while the dominant clones of the thermal water were affiliated with the genus Denitratisoma sp. Most of the identified species and genera were related to bacteria with obligate or facultative chemolithotrophic sulfur metabolism, so the microbes of the curative waters may participate in the sulfur-cycle of the wells.