Enhanced degradation of 4-aminobenzenesulfonate by a co-culture of Afipia sp. 624S and Diaphorobacter sp. 624L.

Two strains, Afipia sp. 624S and Diaphorobacter sp. 624L, were isolated from an enrichment culture with 4-aminobenzenesulfonate (4-ABS) as the only carbon source. Strain 624S utilized 4-ABS as the only source of carbon and energy and degraded 3.8 mM 4-ABS in 2 weeks, releasing a small amount of sulfate ions. On the other hand, strain 624L did not utilize 4-ABS. Additionally, a co-culture of strains 624S and 624L resulted in the enhanced degradation of 4-ABS, and no sulfite was accumulated in the degradation of 4-ABS. When incubated in 50 mM Tris-HCl buffer (pH 8.0) containing 2.2 mM sodium sulfite, strain 624S exhibited no sulfite oxidation; however, strain 624L completely oxidized the sulfite after 2 days. Furthermore, when manganase, which has the ability to oxidize sulfite, was added to the medium, the degradation rate of 4-ABS was increased in comparison with the non-addition control. These results indicate that the sulfite oxidation might stimulate the degradation of 4-ABS by strain 624S, suggesting syntrophic interaction between strains 624S and 624L based on sulfite oxidation.

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.

Biological wastewater treatment of 1,4-dioxane using polyethylene glycol gel carriers entrapping Afipia sp. D1.

A biological treatment system for 1,4-dioxane-containing wastewater was developed using the bacterium Afipia sp. D1, which can utilize 1,4-dioxane as the sole carbon source. Strain D1 was entrapped in a polyethylene glycol gel carrier to stably maintain it in a bioreactor, and continuous feeding tests were performed to treat model industrial wastewater containing 1,4-dioxane. 1,4-Dioxane removal activity rapidly increased soon after the start of feeding of influent with 400 mg/L 1,4-dioxane, and the volumetric removal rate reached 0.67 kg dioxane/m(3)/d on day 36 by a stepwise increase in loading. The start-up period of the 1,4-dioxane treatment reactor was approximately 1 month, and stable removal performance was subsequently achieved for more than 1 month. The average 1,4-dioxane effluent concentration and 1,4-dioxane removal efficiency were 3.6 mg/L and 99%, respectively, during stable operation. Further 1,4-dioxane degradation activity of the of the gel carrier was characterized in batch experiments with respect to temperature. The optimum temperature for 1,4-dioxane treatment was 31.7°C, and significant removal was observed at a temperature as low as 6.9°C. The apparent activation energy for 1,4-dioxane degradation was estimated to be 47.3 kJ/mol. This is the first report of the development of a 1,4-dioxane biological treatment system using gel entrapment technology.

Pilot test of biological removal of 1,4-dioxane from a chemical factory wastewater by gel carrier entrapping Afipia sp. strain D1.

A pilot-scale (120 L) bioreactor system using a gel carrier-entrapped pure bacterial strain, Afipia sp. strain D1, capable of degrading 1,4-dioxane as a sole carbon and energy source was constructed and applied to treat real industrial wastewater containing 1,4-dioxane from a chemical factory. Although the wastewater not only contained high concentrations of 1,4-dioxane but also considerable amounts of other organic compounds (73 mg-TOCL(-1) on average), the bioreactor could efficiently remove 1,4-dioxane without significant inhibitory effects. The reactor startup could be completed within approximately 1 month by increasing the 1,4-dioxane loading rate (0.09-0.47 kg-dioxanem(-3)d(-1)) in a stepwise manner. Effective 1,4-dioxane removal was stably maintained for 3 months with an influent 1,4-dioxane of 570-730 mg L(-1), giving an average effluent concentration and removal rate of 3.4 mg L(-1) and 0.46 kg-dioxanem(-3)d(-1), respectively. A 1,4-dioxane loading fluctuation between 0.14 and 0.72 kg-dioxanem(-3)d(-1) did not significantly affect its removal, and more than 99% removal efficiency was constantly maintained. The Monod model could well describe the relationship between the effluent 1,4-dioxane concentration and 1,4-dioxane removal rates of the bioreactors, showing that the half-saturation constant (Ks) was 28 mg L(-1).

Identification of a novel Afipia species isolated from an Indian flying fox.

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

Experimental infection of cats with Afipia felis and various Bartonella species or subspecies.

Based upon prior studies, domestic cats have been shown to be the natural reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. However, other Bartonella species, such as Bartonella vinsonii subsp. berkhoffii, Bartonella quintana or Bartonella bovis (ex weissii) have been either isolated from or Bartonella DNA sequences PCR amplified and sequenced. In the late 1980s, before B. henselae was confirmed as the etiological agent of cat scratch disease, Afipia felis had been proposed as the causative agent. In order to determine the feline susceptibility to A. felis, B. vinsonii subsp. berkhoffii, Bartonella rochalimae, B. quintana or B. bovis, we sought to detect the presence of bacteremia and seroconversion in experimentally-inoculated cats. Most of the cats seroconverted, but only the cats inoculated with B. rochalimae became bacteremic, indicating that cats are not natural hosts of A. felis or the other Bartonella species or subspecies tested in this study.

Isolation and characterization of two novel bacteria Afipia cberi and Mesorhizobium hominis from blood of a patient afflicted with fatal pulmonary illness.

We recently isolated and discovered new Bradyrhizobiaceae microbes from the cryopreserved culture broth of blood samples from 3 patients with poorly defined illnesses using modified SP4 media and culture conditions coupled with genomic sequencing. Using a similar protocol, we studied a previously cryopreserved culture broth of blood sample from a patient who had succumbed to an acute onset of fulminant pulmonary illness. We report that two phases of microbial growth were observed in the re-initiated culture. Biochemical and genomic characterization revealed microbes isolated from the first phase of growth were new Afipia species of Bradyrhizobiaceae, tentatively named A. cberi with a ~ 5 MB chromosome that was different from those of all previously known Afipia microbes including the newly discovered A. septicemium. The microbes isolated from the second phase of growth were prominent sugar assimilators, novel Phyllobacteriaceae, phylogenetically most closely related to Mesorhizobium and tentatively named M. hominis with a ~ 5.5 MB chromosome. All A. cberi isolates carry a circular ~ 140 KB plasmid. Some M. hominis isolates possess a circular ~ 412 KB plasmid that can be lost in prolonged culture or passage. No antibiotics resistant genes could be identified in both of the A. cberi and M. hominis plasmids. Antibiotic susceptibility studies using broth culture systems revealed isolates of A. cberi could be sensitive to some antibiotics, but all isolates of M. hominis were resistant to essentially all tested antibiotics. However, the cell-free antibiotics susceptibility test results may not be applicable to clinical treatment against the microbes that are known to be capable of intracellular growth. It remains to be determined if the 2 previously unknown Rhizobiales were indeed pathogenic and played a role in the pulmonary disease process in this patient. Specific probes and methods will be developed to re-examine the diseased lungs from patient's autopsy.

Isolation of novel Afipia septicemium and identification of previously unknown bacteria Bradyrhizobium sp. OHSU_III from blood of patients with poorly defined illnesses.

Cultures previously set up for isolation of mycoplasmal agents from blood of patients with poorly-defined illnesses, although not yielding positive results, were cryopreserved because of suspicion of having low numbers of unknown microbes living in an inactive state in the broth. We re-initiated a set of 3 cultures for analysis of the "uncultivable" or poorly-grown microbes using NGS technology. Broth of cultures from 3 blood samples, submitted from OHSU between 2000 and 2004, were inoculated into culture flasks containing fresh modified SP4 medium and kept at room temperature (RT), 30°C and 35°C. The cultures showing evidence of microbial growth were expanded and subjected to DNA analysis by genomic sequencing using Illumina MiSeq. Two of the 3 re-initiated blood cultures kept at RT after 7-8 weeks showed evidence of microbial growth that gradually reached into a cell density with detectable turbidity. The microbes in the broth when streaked on SP4 agar plates produced microscopic colonies in ∼ 2 weeks. Genomic studies revealed that the microbes isolated from the 2 blood cultures were a novel Afipia species, tentatively named Afipia septicemium. Microbes in the 3(rd) culture (OHSU_III) kept at RT had a limited level of growth and could not reach a plateau with high cell density. Genomic sequencing identified the microbe in the culture as a previously unknown species of Bradyrhizobium bacteria. This study reports on the isolation of novel Afipia and Bradyrhizobium species. Isolation of Bradyrhizobium species bacteria has never been reported in humans. The study also reveals a previously unrecognized nature of hematogenous infections by the 2 unique groups of Bradyrhizobiaceae. Our studies show that improvement of culture system plus effective use of NGS technology can facilitate findings of infections by unusual microbes in patients having poorly-defined, sometimes mysterious illnesses.

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.

Amoebal pathogens as emerging causal agents of pneumonia.

Despite using modern microbiological diagnostic approaches, the aetiological agents of pneumonia remain unidentified in about 50% of cases. Some bacteria that grow poorly or not at all in axenic media used in routine clinical bacteriology laboratory but which can develop inside amoebae may be the agents of these lower respiratory tract infections (RTIs) of unexplained aetiology. Such amoebae-resisting bacteria, which coevolved with amoebae to resist their microbicidal machinery, may have developed virulence traits that help them survive within human macrophages, i.e. the first line of innate immune defence in the lung. We review here the current evidence for the emerging pathogenic role of various amoebae-resisting microorganisms as agents of RTIs in humans. Specifically, we discuss the emerging pathogenic roles of Legionella-like amoebal pathogens, novel Chlamydiae (Parachlamydia acanthamoebae, Simkania negevensis), waterborne mycobacteria and Bradyrhizobiaceae (Bosea and Afipia spp.).

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.

Biogenesis of Afipia-containing phagosomes in non-professional phagocytes.

Afipia felis is a Gram-negative alpha-proteobacterium, a rare cause of human cat scratch disease (CSD), and likely a pathogen of amoeba. Here, we show that various members of the genus Afipia attach to and are taken up by various non-professional phagocytic mammalian cells (epithelial CHO, endothelial EA.hy926, epithelial HeLa, epithelial INT407 cells, endothelial HMEC-1, endothelial HUVEC, and fibroblast L929 cells). However, only A. felis was able to do this efficiently. Invasion depended on a functional actin cytoskeleton and much less on microtubule dynamics. Bacteria were slowly taken up into HMEC-1 (and HUVEC) via pocket-like structures and they resided within membrane-surrounded phagosomes. While A. felis was found in a non-canonical endocytic compartment in macrophage cells, Afipia-containing phagosomes in HMEC-1 were transiently positive for early endosomal EEA1 and then became and remained positive for lysosome-associated membrane protein-1 (LAMP1) and the proton-pumping ATPase, suggesting undisturbed, albeit slowed, phagosome biogenesis in these cells. Similarly, at 24h of infection, most phagosomes in HeLa, INT407, HUVEC and in EA.hy926 cells were positive for LAMP1. In summary, A. felis enters various non-professional phagocytes and its compartmentation differs between macrophages and non-professional phagocytes.

Lipid microdomain-dependent macropinocytosis determines compartmentation of Afipia felis.

Phagocytic compartments are specialized endocytic organelles and usually mature along the degradative pathway into phagolysosomes. The rare human pathogen Afipia felis localizes to a compartment that is different from canonical phagocytic compartments. Here, we present evidence that internalization of Afipia by macrophages and unusual phagosome development are considerably decreased by attachment of cholera toxin B subunit to macrophage ganglioside GM1 or by extraction or oxidation of plasma membrane cholesterol. Amiloride (an inhibitor of Na(+)/H(+) exchanger and macropinocytosis) strongly inhibited uptake of A. felis at a late step, i.e. the closure of macropinocytic structures rather than the production of membrane ruffles. Ultrastructural evidence showed that A. felis was taken up by macrophages via macropinocytosis. In contrast, A. felis opsonized with a monoclonal IgG antibody was ingested by a zipper-like mechanism, resulting in normal phagosome maturation. Hence, while the preferred path of A. felis uptake is dependent on the integrity of lipid microdomains and on macropinocytosis, and while this uptake leads to an unusual phagosome and to intracellular survival of A. felis, those bacteria that enter using Fcgamma receptors are delivered to a late endocytic compartment.

[Cat-scratch disease as cause of Lymphadenitis colli]. / Katzenkratzkrankheit als Ursache einer Lymphadenitis colli.

Cat-scratch Disease as Cause of Lymphadenitis colli. Cat-scratch disease is a frequent cause of lymphadenitis colli. It mainly affects children and adolescents younger than 21 years. Since the clinical picture is not characteristic, a history of contact to cats or kittens is highly valuable for diagnosing the disease. Major aspects of the disease concerning epidemiology, diagnostic procedures, clinical presentation and therapy are discussed.

PET scan-positive cat scratch disease in a patient with T cell lymphoblastic lymphoma.

In patients who have history of lymphoma, a positive positron emission tomography (PET) scan is frequently considered as good evidence for relapse and/or persistent disease. Thus, lymph node biopsy is not always done to confirm the diagnosis of relapse or refractory lymphoma before a patient is subjected to further chemotherapy. We report a case of patient with history of T cell lymphoblastic lymphoma who presented again with inguinal lymphadenopathy and positive study on positron emission tomography suggestive of lymphoma relapse. This was pathologically proven to be cat scratch disease. This case suggests that in the immunocompromised patients who had history of lymphoma, infectious etiology should be ruled out for PET scan-positive lymphadenopathy.

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.