Rickettsia monacensis sp. nov., a spotted fever group Rickettsia,from ticks (Ixodes ricinus) collected in a European city park

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.     

Spotted fever group Rickettsia in ticks from southeastern Spain natural parks

During an 8-years study, we collected from vegetation or domestic and wild mammals 1246 ticks (624 males, 511 females and 111 nymphs) belonging to 13 species in Jaen province (Andalusia) and we analyzed these ticks by PCR and sequencing for the presence of rickettsiae. Specific rickettsiae DNA was detected in 243 (19.5%) of the ticks tested. Sequence analysis of amplicons of gltA, ompA and ompB genes revealed that Ixodes ricinus were infected with R. monacensis, including strain IRS3, and R. helvetica (prevalences of 27.0% and 2.7%, respectively), while in I. ventalloi we found only this last species (12.5%). Moreover, Dermacentor marginatus presents R. slovaca (24.7%) and R. raoultii (59.9%). In Rhipicephalus sanguineus group ticks (Rh. sanguineus, Rh. turanicus and Rh. pusillus) only R. massiliae (15.2%) was found. Haemaphysalis punctata and Ha. sulcata were infected with a Rickettsia sp. near R. hoogstraalii (prevalence of 3.1% and 16.1%, respectively). In addition, Ha. punctata appeared infected with R. monacensis-like Rickettsia (1.0%) and R. raoultii (9.3%). None of I. hexagonus, Hyalomma lusitanicum, Hyalomma sp., Ha. hispanica or Rh. bursa studied ticks contained rickettsiae.     

Phylogenetic Analysis of Phenotypically Characterized Cryptococcus laurentii Isolates Reveals High Frequency of Cryptic Species

Background

Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States.

Methods

In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region.

Results

BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99–100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified.

Conclusions

Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.     

Rickettsia monteiroi sp. nov., infecting the tick Amblyomma incisum in Brazil

Free-living adult Amblyomma incisum ticks were collected in an Atlantic rainforest area at Intervales State Park, State of São Paulo, Brazil. From an A. incisum specimen, rickettsiae were successfully isolated in Vero cell culture by the shell vial technique. Rickettsial isolation was confirmed by optical microscopy, transmission electron microscopy, and PCRs targeting portions of the rickettsial genes gltA, htrA, rrs, and sca1 on infected cells. Fragments of 1,089, 457, 1,362, and 443 nucleotides of the gltA, htrA, rrs, and sca1 genes, respectively, were sequenced. By BLAST analysis, the partial sequence of rrs of the A. incisum rickettsial isolate was closest to the corresponding sequence of Rickettsia bellii (99.1% similarity). The gltA partial sequence was closest to the corresponding sequences of “Candidatus Rickettsia tarasevichiae” (96.1% similarity) and Rickettsia canadensis (95.8% similarity). The htrA partial sequence was closest to the corresponding sequence of R. canadensis (89.8% similarity). The sca1 partial sequence was closest to the corresponding sequence of R. canadensis (95.2% similarity). Since our rickettsial isolate was genetically distinct from other Rickettsia species, we propose a new species designated Rickettsia monteiroi sp. nov. Phylogenetic analyses indicated that R. monteiroi belongs to the canadensis group within the genus Rickettsia, together with the species R. canadensis and “Candidatus R. tarasevichiae”. Little or no antibody cross-reaction was observed between sera of R. monteiroi-inoculated guinea pigs and R. bellii-, Rickettsia rickettsii-, or R. canadensis-inoculated guinea pigs.     

Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.     

Rickettsia spp., Ehrlichia sp. and Candidatus Midichloria sp. associated to ticks from a protected urban area in Buenos Aires City (Argentina)

The aim of this study was to determine the infection with Rickettsiales in ticks and birds from the main protected urban area of Buenos Aires City (Argentina). One Amblyomma aureolatum (0.2%) and one Ixodes auritulus (0.1%) were positive by PCR targeting Rickettsia 23S-5S rRNA intergenic spacer. Phylogenetic analysis shows to findings in A. aureolatum are closely to Rickettsia bellii and for I. auritulus are related to ‘Candidatus Rickettsia mendelii’. One I. auritulus (0.1%) and three A. aureolatum (0.6%) were positive by PCR for a fragment of the 16S rRNA gene of the Anaplasmataceae family. The sequences obtained from A. aureolatum were phylogenetically related to Midichloriaceae endosymbionts. The sequence from I. auritulus s.l. had 100% identity with Ehrlichia sp. Magellanica from Chile and two genotypes of Ehrlichia sp. from Uruguay. The results of our study show that Rickettsia and Ehrlichia are present in ticks in the main protected urban area of Buenos Aires City.

     

Development of shuttle vectors for transformation of diverse Rickettsia species

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.     

Metal-characterization of N-Acyl-D-glutamate Amidohydrolase from Pseudomonas sp. Strain 5f-l

N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was a zinc-metalloenzyme which contained 2.06–2.61 g. atom of Zn per mole of enzyme. The zinc atom was required for the catalytic activity and stability of the enzyme. The N-terminal amino acid sequence of Pseudomonas sp. 5f-l D-AGase showed 32% identity to that of Alcaligenes xylosoxydans subsp. xylosoxydans A-6.     

Molecular Evidence for the Presence of Rickettsia Felis in the Feces of Wild-living African Apes

Background

Rickettsia felis is a common emerging pathogen detected in mosquitoes in sub-Saharan Africa. We hypothesized that, as with malaria, great apes may be exposed to the infectious bite of infected mosquitoes and release R. felis DNA in their feces.

Methods

We conducted a study of 17 forest sites in Central Africa, testing 1,028 fecal samples from 313 chimpanzees, 430 gorillas and 285 bonobos. The presence of rickettsial DNA was investigated by specific quantitative real-time PCR. Positive results were confirmed by a second PCR using primers and a probe targeting a specific gene for R. felis. All positive samples were sequenced.

Results

Overall, 113 samples (11%) were positive for the Rickettsia-specific gltA gene, including 25 (22%) that were positive for R. felis. The citrate synthase (gltA) sequence and outer membrane protein A (ompA) sequence analysis indicated 99% identity at the nucleotide level to R. felis. The 88 other samples (78%) were negative using R. felis-specific qPCR and were compatible with R. felis-like organisms.

Conclusion

For the first time, we detected R. felis in wild-living ape feces. This non invasive detection of human pathogens in endangered species opens up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases, beside HIV and malaria.     

Emerging rickettsioses

Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.     

Molecular phylogeny of the marine <Emphasis Type="Italic">Prasiola</Emphasis> and <Emphasis Type="Italic">Rosenvingiella</Emphasis> species (Chlorophyta: Prasiolales) from southeastern Kamchatka

Molecular phylogenetic tools are often useful in distinguishing cryptic species with similar morphologies, but they can also be helpful in identifying morphotypes of a species, which displays completely different shapes. We performed molecular-phylogenetic analysis of supra-tidal green algae in the Kamchatka peninsula that belong to the order Prasiolales (Trebouxiophyceae, Chlorophyta). Based on rbcL sequences results, two new species were recorded for the first time in Kamchatka. Approximately 1.4% of the field-collected Rosenvingiella constricta had a unique uniseriate hood-like blade shape, although their rbcL sequences were 100% identical with typical multiseriate filamentous plants. Kamchatka’s population of R. constricta showed 99.4–99.6% identity in rbcL sequences with the populations from Canada and New Zealand. Another similar-looking Rosenvingiella species collected from the same locality had 93.5% identity of the rbcL gene sequence with R. constricta. Morphological and geographical analyses also suggested that this species might be a new species of the genus Rosenvingiella. Prasiola delicata was recorded for the first time in Kamchatka. The Kamchatka population of P. delicata showed 100% identity in rbcL gene sequence with the population from Vancouver, but differed from the Canadian population morphologically.     

Biodegradation of beta-cyfluthrin by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain S1

-Cyfluthrin [-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like -cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade -cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading -cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of -cyfluthrin have been identified as -cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy--cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).     

<Emphasis Type="Italic">Flavobacterium knui</Emphasis> sp. nov., a novel member of the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>

A Gram-stain negative, non-motile, rod-shaped bacterial strain, designated 2-56^T, was isolated from water and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 2-56^T belongs to the family Flavobacteriaceae in the phylum Bacteroidetes and is closely related to Flavobacterium paronense KNUS1^T (98.4%) and Flavobacterium collinsense 4-T-2^T (96.7%). The G?+?C content of the genomic DNA of strain 2-56^T was 33.4 mol%. The isolate contained MK-6 as the predominant respiratory quinone, and iso-C_15:1 G (15.9%), iso-C_15:0 (15.8%), iso-C_17:0 3-OH (10.7%), and iso-C_15:0 3-OH (9.6%) were the major fatty acids. The major polar lipids were phosphatidylethanolamine and an unidentified lipid. The phenotypic and chemotaxonomic data support the affiliation of strain 2-56^T with the genus Flavobacterium. However, the DNA–DNA relatedness between the isolate and F. paronense and F. collinsense were 35.7 and 21.5%, respectively, clearly showing that strain 2-56^T is not related to them at the species level. Strain 2-56^T could be clearly differentiated from its close neighbours on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain 2-56^T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium knui sp. nov. is proposed. The type strain is 2-56^T (=?KCTC 62061^T?=?JCM 32247^T).     

<Emphasis Type="Italic">Abyssisolibacter fermentans</Emphasis> gen. nov. sp. nov., isolated from deep sub-seafloor sediment

A Gram-staining-negative, thin rod-shaped, anaerobic bacterium designated MCWD3^T was isolated from sediment of the deep sea in Ulleung Basin, East Sea, Korea. The ranges of temperature, pH and NaCl for growth of this strain were 15–40°C (optimum 29°C), 5.0–10.0 (optimum pH 6.5), and 1–5%, respectively. The major fatty acids were iso-C_15:0 (30%) and iso-C_15:0 dimethyl acetal (17%). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified aminophospholipids, phospholipids, and aminolipids. The fermentation product from yeast extract was acetate. Phylogenetic analysis based on 16S rRNA genes indicated that the isolate was related to Sporosalibacterium faouarense (92.8% sequence identity), Clostridiisalibacter paucivorans (92.6%), and Brassicibacter mesophilus (92.4%). However, the isolate was differentiated from these genera by both physiological and chemotaxonomical properties. On the basis of a polyphasic taxonomic analysis, we propose that MCWD3^T represents a novel taxon with the name Abyssisolibacter fermentans gen. nov. sp. nov.     

Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: a model system

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Rapid and simple purification of a novel extracellular β-amylase from Bacillus sp.

Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M _r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-.     

Antrihabitans stalagmiti sp. nov., isolated from a larva cave and a proposal to transfer Rhodococcus cavernicola Lee et al. 2020 to a new genus Spelaeibacter as Spelaeibacter cavernicola gen. nov. comb. nov

An actinobacterial strain, designated YC3-6^T, was isolated from a larva cave in Jeju, Republic of Korea. The novel isolate was found to grow at 10–30 °C, pH 5.0–10.0 and 0–4% (w/v) NaCl. The 16S rRNA gene phylogeny showed that the novel isolate formed a distinct subline within the family Nocardiaceae. Levels of 16S rRNA gene similarity indicated that the close relatives are Rhodococcus cavernicola (98.4% sequence similarity) and “Rhodococcus psychrotolerans” (98.2%) followed by Antrihabitans stalactiti (96.8%). However, the core gene-based phylogeny revealed that the novel isolate formed a tight cluster with A. stalactiti and was separated from R. cavernicola and other members of the family Nocardiaceae. The morphological and chemotaxonomic characteristics of strain YC3-6^T are in line with those of the genus Antrihabitans. Strain YC3-6^T showed an average nucleotide identity of 75.5% and a digital DDH of 20.3% with A. stalactiti. In addition, the core gene analysis showed that R. cavernicola formed a distinct subline between an Antrihabitans cluster and Aldersonia kunmingensis, and well separated from members of the genus Rhodococcus. The average amino acid identity values of R. cavernicola to closely related neighbours were 69.3–69.4% with members of the genus Antrihabitans and 67.3% with Ald. kunmingensis, while the POCP values ranged from 56.9 to 63.6%. On the basis of results obtained here, strain YC3-6^T is concluded to represent a novel species of the genus Antrihabitans, for which the name Antrihabitans stalagmiti sp. nov. (type strain, YC3-6^T?=?KACC 19963^T?=?DSM 107561^T) is proposed. Based on overall genome relatedness and chemotaxonomic differences, it is also proposed that R. cavernicola Lee et al. 2020 be transferred to a new genus Spelaeibacter as Spelaeibacter cavernicola gen. nov., comb. nov.

     

Prevalence of Rickettsia and Bartonella species in Spanish cats and their fleas

The aim of this study was to determine the prevalence of Bartonella henselae, Rickettsia felis, and Rickettsia typhi in fleas and companion cats (serum and claws) and to assess their presence as a function of host, host habitat, and level of parasitism. Eighty‐nine serum and claw samples and 90 flea pools were collected. Cat sera were assayed by IFA for Bartonella henselae and Rickettssia species IgG antibodies. Conventional PCRs were performed on DNA extracted from nails and fleas collected from cats. A large portion (55.8%) of the feline population sampled was exposed to at least one of the three tested vector‐borne pathogens. Seroreactivity to B. henselae was found in 50% of the feline studied population, and to R. felis in 16.3%. R. typhi antibodies were not found in any cat. No Bartonella sp. DNA was amplified from the claws. Flea samples from 41 cats (46%) showed molecular evidence for at least one pathogen; our study demonstrated a prevalence rate of 43.3 % of Rickettsia sp and 4.4% of Bartonella sp. in the studied flea population. None of the risk factors studied (cat's features, host habitat, and level of parasitation) was associated with either the serology or the PCR results for Bartonella sp. and Rickettsia sp.. Flea‐associated infectious agents are common in cats and fleas and support the recommendation that stringent flea control should be maintained on cats.