Draft genome sequence of "Candidatus Afipia apatlaquensis" sp. nov., IBT-C3, a potential strain for decolorization of textile dyes.

OBJECTIVES: In order to characterize a river-associated, enriched microbiome capable of degrading an anthraquinone dye from the oil blue family, as well as assessing its functional potential, we performed a taxa-specific metagenomic deconvolution analysis based on contact probability maps at the chromosomal level. This study will allow associating the genomic content of "Candidatus Afipia apatlaquensis" strain IBT-C3 with its phenotypic potential in the context of bioremediation of textile dyes. We anticipate that this resource will be very useful in comparative genomic clinical studies, contributing to understanding the genomic basis of Afipia pathogenicity. DATA DESCRIPTION: Here, we report the first draft genome sequence of "Candidatus Afipia apatlaquensis" sp. nov., strain IBT-C3, obtained by deconvolution of a textile-dye degrader microbiome in Mexico. The genome composite was deconvoluted using a Hi-C proximity ligation method. Whole-genome-based comparisons and phylogenomics reconstruction indicate that strain IBT-C3 represents a new species of the genus Afipia. The assembly completeness was 92.5% with 5,604,749 bp in length and 60.72% G+C content. The genome complement of IBT-C3 suggests a functional potential for decolorization of textile dyes, contrasting with previous reports of Afipia genus focused on its pathogenic potential.

Degradation of methomyl by the combination of Aminobacter sp. MDW-2 and Afipia sp. MDW-3.

Methomyl (S-methyl N-(methylcarbamoyloxy) thioacetimidate) is a kind of oxime carbamate insecticide. It is considered to be extremely toxic to nontarget organism. To date, no pure culture or consortium has been reported to have the ability to degrade methomyl completely. In this study, a methomyl-degrading enrichment E1 was obtained by using the sludge from the wastewater-treating system of a pesticide manufacturer as the original inoculant. Two bacterial strains named MDW-2 and MDW-3 were isolated from this enrichment, and they were preliminarily identified as Aminobacter sp. and Afipia sp. respectively. Strains MDW-2 and MDW-3 could coexist and degrade 50 mg l-1 methomyl completely within 3 days by the cooperative metabolism. Methomyl was first converted to methomyl oxime and methylcarbamic acid by strain MDW-2, and the latter could be used as the carbon source for the growth of strain MDW-2. But methomyl oxime could not be sequentially degraded by strain MDW-2. However, it could be degraded and used as the carbon source by strain MDW-3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a bacterial combination of Aminobacter sp. MDW-2 and Afipia sp. MDW-3, which could degrade methomyl completely by biochemical cooperation. This study also proposes the biodegradation pathway of methomyl for the first time and highlights the application potential of a bacterial combination in the remediation of methomyl-contaminated environments.

Genome sequence of Afipia birgiae, a rare bacterium associated with Amoebae.

Afipia birgiae is an alphaproteobacterium from the family Bradyrhizobiaceae, growing in amoebae, and a potential human pathogen. We sequenced the genome of type strain 34632(T). It is composed of 5,325,467 bp and contains 5,160 protein-coding genes and 53 RNA genes, including 3 rRNA genes.

Haloacetic acid-degrading bacterial communities in drinking water systems as determined by cultivation and by terminal restriction fragment length polymorphism of PCR-amplified haloacid dehalogenase gene fragments.

AIMS: To characterize the HAA-degrading bacteria in drinking water systems. METHODS AND RESULTS: Haloacetic acid (HAA)-degrading bacteria were analysed in drinking water systems by cultivation and by a novel application of terminal restriction fragment length polymorphism (tRFLP). Substantial similarities were observed among the tRFLP patterns of dehI and dehII gene fragments in drinking water samples obtained from three different cities (Minneapolis, MN; St Paul, MN; Bucharest, Romania) and from one biologically active granular activated carbon filter (Hershey, PA). The dominant fragment in the tRFLP profiles of dehI genes from the drinking water samples matched the pattern from an Afipia sp. that was previously isolated from drinking water. In contrast, the dominant fragment in the tRFLP profiles of dehII genes did not match any previously characterized dehII gene fragment. PCR cloning was used to characterize this gene fragment, which had <65% nucleotide sequence identity with any previously characterized dehII gene. CONCLUSIONS: Afipia spp. are an appropriate model organism for studying the biodegradation of HAAs in drinking water distribution systems as encoded by dehI genes; the organism that harbours the most prominent dehII gene in drinking water has yet to be cultivated and identified. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a novel application of tRFLP targeting dehI and dehII genes could be broadly useful in understanding HAA-degrading bacteria in numerous environments.

The Afipia toolbox and its use to isolate flagellar mutants.

Afipia felis, a Gram-negative alphaproteobacterium, has been implicated as one of the causative agents of cat scratch disease. To identify and begin to examine the virulence traits of this organism, we developed and tested a highly efficient transposon delivery system and a stable plasmid vector expressing green fluorescent protein. The transposome system is based on a Tn5-derived transposon and a phage restriction endonuclease type I inhibitor. Electroporation of this construct produced a library of >2600 mutants, which were screened for flagella biosynthesis mutants using a monoclonal antibody to Afipia flagellin. Insertion loci for two selected mutants were located in the genes for flagellin and flagellin biosynthesis FlhA, confirming the validity of the approach.

Isolation and characterization of haloacetic acid-degrading Afipia spp. from drinking water.

Haloacetic acids are a class of disinfection byproducts formed during the chlorination and chloramination of drinking water that have been linked to several human health risks. In this study, we isolated numerous strains of haloacetic acid-degrading Afipia spp. from tap water, the wall of a water distribution pipe, and a granular activated carbon filter treating prechlorinated water. These Afipia spp. harbored two phylogenetically distinct groups of alpha-halocarboxylic acid dehalogenase genes that clustered with genes previously detected only by cultivation-independent methods or were novel and did not conclusively cluster with the previously defined phylogenetic subdivisions of these genes. Four of these Afipia spp. simultaneously harbored both the known classes of alpha-halocarboxylic acid dehalogenase genes (dehI and dehII), which is potentially of importance because these bacteria were also capable of biodegrading the greatest number of different haloacetic acids. Our results suggest that Afipia spp. have a beneficial role in suppressing the concentrations of haloacetic acids in tap water, which contrasts the historical (albeit erroneous) association of Afipia sp. (specifically Afipia felis) as the causative agent of cat scratch disease.

Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes.

AIMS: To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs). METHODS AND RESULTS: Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify dehII genes from selected indicator strains. The developed primer sets were effective in directly amplifying dehII genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the dehII gene was used. CONCLUSIONS: This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify dehII genes in drinking water systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.

Isolation and properties of methanesulfonate-degrading Afipia felis from Antarctica and comparison with other strains of A. felis.

Three novel strains of methylotrophic Afipia felis were isolated from several locations on Signy Island, Antarctica, and a fourth from estuary sediment from the River Douro, Portugal. They were identified as strains of the alpha-2 proteobacterium A. felis by 16S rRNA gene sequence analysis. Two strains tested were shown to contain the fdxA gene, diagnostic for A. felis. All strains grew with methanesulfonate (and two strains with dimethylsulfone) as sole carbon substrate. Growth on methanesulfonate required methanesulfonate monooxygenase (MSAMO), using NADH as the reductant and stimulated by reduced flavin nucleotides and Fe(II). Polymerase chain reaction amplification of DNA from an Antarctic strain showed a typical msmA gene for the alpha-hydroxylase of MSAMO, and both Antarctic and Portuguese strains contained mxaF, the methanol dehydrogenase large subunit gene. This is the first report of methanesulfonate-degrading bacteria from the Antarctic and of methylotrophy in Afipia, and the first description of any bacterium able to use both methanesulfonate and dimethylsulfone. In contrast, the type strain of A. felis DSM 7326(T) was not methylotrophic, but grew in defined mineral medium with a wide range of single simple organic substrates. Free-living Afipia strains occurring widely in the natural environment may be significant as methylotrophs, degrading C(1)-sulfur compounds, including the recalcitrant organosulfur compound methanesulfonate.

Rare bacterium of new genus isolated with prolonged enrichment culture.

Dynamic change in microbial flora was monitored with an oxygen electrode. The 1st phase microorganisms, which first grew well in LB medium, were followed by the 2nd phase microorganisms, which supposedly assimilated microbial cells of the 1st phase and their metabolites. In a similar way, a change in microbial flora was observed from the 1st phase to the 4th phase in 84 hr. Based on this observation, prolonged enrichment culture was done for as long as two months to increase the ratio of existence of rare microorganisms. From these culture liquids, four slow-growing bacteria (provisionally named Shinshu-ah1, -ah2, -ah3, and -ah4), which formed scarcely visible small colonies, were isolated. Sequence analysis of their 16S rDNA showed that Shinshu-ah1 had 97% homology with Bradyrhizobium japonicum and uncultured alpha proteobacterium clone blaii 16, Shinshu-ah2 91% with Rasbo bacterium, Alpha proteobacterium 34619, Bradyrhizobium genosp. P, Afipia felis and an unidentified bacterium, Shinshu-ah3 99% with Methylobacterium mesophilicum, and Shinshu-ah4 95% with Agromyces ramosus DSM 43045. Phylogenetic study indicated that Shinshu-ah2 had a possibility to form a new family, Shinshu-ah1 a new genus, and Shinshu-ah4 a new species.

Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences.

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

Rapid polymerase chain reaction-based confirmation of cat scratch disease and Bartonella henselae infection.

CONTEXT: Cat scratch disease (CSD) commonly occurs secondary to Bartonella henselae infection, and the diagnosis has traditionally been made by microscopic findings, the identification of organisms by cytochemistry, and clinical history. However, cytochemical analysis tends to be very difficult to interpret, and histology alone may be insufficient to establish a definitive diagnosis of CSD. OBJECTIVE: To demonstrate the presence of B henselae in tissue suspected of involvement by CSD, using a novel polymerase chain reaction (PCR) assay. DESIGN: Isolates of B henselae (American Tissue Culture Collection 49793) and Afipia felis (American Tissue Culture Collection 49714) were cultured on blood agar and buffered charcoal yeast extract agar, respectively. DNA was isolated from these organisms and from formalin-fixed, paraffin-embedded tissue sections with involvement by CSD (8 patients). Negative controls included water, human placental tissue, and lymph node specimens from 6 patients with reactive lymphoid hyperplasia and from 2 patients with granulomatous lymphadenitis. A primer complementary to B henselae citrate synthase gltA gene sequence was designed to perform a seminested PCR amplification. For restriction fragment length polymorphism analysis, PCR products were digested by TaqI restriction enzyme and analyzed by gel electrophoresis. RESULTS: Seminested PCR analysis of the cultured isolates of B henselae, but not of A felis, showed specific amplification. However, nonnested PCR did not provide consistently positive results in tissue sections with CSD. Therefore, we used a seminested PCR, which revealed positivity in all of the cases with clinicopathologic diagnoses of CSD. None of the negative controls showed positivity. Restriction enzyme provided confirmation of the specific PCR amplification of the B henselae sequence. CONCLUSIONS: Since the amplification product has a low molecular size (<200 base pairs), this assay is useful for detection of B henselae in formalin-fixed, paraffin-embedded tissues. The seminested PCR protocol described here can be used for rapid and reliable confirmation of B henselae in samples that are histologically suggestive of CSD.

Description of Afipia birgiae sp. nov. and Afipia massiliensis sp. nov. and recognition of Afipia felis genospecies A.

On the basis of phenotypic characterization and DNA relatedness, two novel species are proposed, Afipia birgiae sp. nov. (type strain 34632T = CIP 106344T = CCUG 43108T) and Afipia massiliensis sp. nov. (type strain 34633T = CIP 107022T = CCUG 45153T). A new genospecies is described, named Afipia felis genospecies A, closely related to Afipia felis. The complexity encountered in the taxonomy of the Bradyrhizobiaceae group within the alpha-2 subgroup of the Proteobacteria is discussed and the description of these novel species highlights the need for new tools for phylogenetic analysis in the group. The novel species herein described are fastidious bacteria isolated from a hospital water supply in co-culture with amoebae. It is hypothesized that this group of bacteria are a potential cause of nosocomial infections.

Detection of Bartonella henselae and Afipia felis DNA by polymerase chain reaction in specimens from patients with cat scratch disease.

Polymerase chain reaction (PCR) amplification and colorimetric identification of amplicons were performed to detect Bartonella henselae and Afipia felis DNA in specimens from patients who were clinically and histologically suspected of having cat scratch disease. PCR products were revealed using 2% ethidium bromide agarose-gel electrophoresis and identified with specific probes in a commercial colorimetric hybridization assay (DEIA) (GEN-ETI-K; DiaSorin, Italy). Six paraffin-embedded lymph node biopsies from 18 patients as well as 18 samples of peripheral whole blood and 18 sera were investigated. Bartonella henselae DNA was recovered from the whole blood of four patients, and Bartonella henselae and Afipia felis DNA were detected in one patient's lymph node biopsy. This study suggests that PCR-DEIA is sufficiently sensitive to be considered feasible for the molecular diagnosis of cat scratch disease.