First report and whole-genome sequencing of Pseudochrobactrum saccharolyticum in Latin America.

The Brucellaceae family comprises microorganisms similar both phenotypically and genotypically, making it difficult to identify the etiological agent of these infections. This study reports the first isolation, identification, and characterization of Pseudochrobactrum saccharolyticum (strain 115) from Latin America. Strain 115 was isolated in 2007 from a bovine in Brazil and was initially classified as Brucella spp. by classical microbiological tests and bcsp31 PCR. The antimicrobial susceptibility of strain 115 was tested against drugs used to treat human brucellosis by minimal inhibitory concentration test. Subsequently, the whole genome of the strain was sequenced, assembled, and characterized. Phylogenetic trees built from 16S rRNA and recA gene sequences enabled the classification of strain 115 as Pseudochrobactrum spp. Phylogenomic analysis using Single Nucleotide Polymorphisms and Average Nucleotide Identity allowed the classification of the strain as P. saccharolyticum. Additionally, a Tetra Correlation Search identified one related genome from the same species, which was compared with strain 115 by analyzing genomic islands. This is the first identification and whole-genome sequence of P. saccharolyticum in Latin America and highlights a challenge in the diagnosis of bovine brucellosis, which could be solved by including the sequencing of 16S rRNA and recA genes in routine diagnostics.

Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts.

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.

Alterations of lung microbiota in a mouse model of LPS-induced lung injury.

Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents.

Enhancement of phenol biodegradation by Pseudochrobactrum sp. through ultraviolet-induced mutation.

The phenol-degrading efficiency of Pseudochrobactrum sp. was enhanced by ultraviolet (UV) irradiation. First, a bacterial strain, Pseudochrobactrum sp. XF1, was isolated from the activated sludge in a coking plant. It was subjected to mutation by UV radiation for 120 s and a mutant strain with higher phenol-degrading efficiency, Pseudochrobactrum sp. XF1-UV, was selected. The mutant strain XF1-UV was capable of degrading 1800 mg/L phenol completely within 48 h and had higher tolerance to hydrogen ion concentration and temperature variation than the wild type. Haldane's kinetic model was used to fit the exponential growth data and the following kinetic parameters were obtained: µmax = 0.092 h-1, Ks = 22.517 mg/L, and Ki = 1126.725 mg/L for XF1, whereas µmax = 0.110 h-1, Ks = 23.934 mg/L, and Ki = 1579.134 mg/L for XF1-UV. Both XF1 and XF1-UV degraded phenol through the ortho-pathway; but the phenol hydroxylase activity of XF1-UV1 was higher than that of XF1, therefore, the mutant strain biodegraded phenol faster. Taken together, our results suggest that Pseudochrobactrum sp. XF1-UV could be a promising candidate for bioremediation of phenol-containing wastewaters.

Paenochrobactrum pullorum sp. nov. isolated from a chicken.

A Gram-stain-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (strain 280(T)) isolated from a chicken was studied for its taxonomic allocation. 16S rRNA gene sequence analyses clearly allocated the isolate in the genus Paenochrobactrum group with a 16S rRNA gene sequence similarity of 98.8% to the currently recognized species, Paenochrobactrum gallinarii and Paenochrobactrum glaciei. This allocation was confirmed by the fatty acid data (major fatty acids: C18:1ω7c and C19:0 cyclo ω8c) and a polyamine pattern with the major compound putrescine and relatively high amounts of spermidine. Also, the polar lipid profile with phosphatidylethanolamine, phosphatiylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine and the genus-specific 'stretched aminolipid' was well in line with the description of the genus Paenochrobactrum. The quinone system consisted predominantly of ubiquinone Q-10 with traces of Q-9 and Q-11. DNA-DNA hybridization of strain 280T with Paenochrobactrum gallinarii Sa25T and Paenochrobactrum glaciei KMM 3858T showed relatedness values of 38.8% (reciprocal 20.2%) and 30.2% (reciprocal 29.8%), respectively. These results in combination with differentiating physiological and biochemical data clearly showed that strain 280T merits species status. We propose the name Paenochrobactrum pullorum sp. nov. to accommodate this strain with the type strain 280T (=LMG 28095T=CIP 110700T).

Comparative phylogenomics and evolution of the Brucellae reveal a path to virulence.

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as "stealth pathogens" that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing "atypical" strains and a highly conserved "classical" core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.

Cr(VI) resistance and removal by indigenous bacteria isolated from chromium-contaminated soil.

The removal of toxic Cr(VI) by microorganisms is a promising approach for Cr(VI) pollution remediation. In the present study, four indigenous bacteria, named LY1, LY2, LY6, and LY7, were isolated from Cr(VI)-contaminated soil. Among the four Cr(VI)-resistant isolates, strain LY6 displayed the highest Cr(VI)-removing ability, with 100 mg/l Cr(VI) being completely removed within 144 h. It could effectively remove Cr(VI) over a wide pH range from 5.5 to 9.5, with the optimal pH of 8.5. The amount of Cr(VI) removed increased with initial Cr(VI) concentration. Data from the time-course analysis of Cr(VI) removal by strain LY6 followed first-order kinetics. Based on the 16S rRNA gene sequence, strain LY6 was identified as Pseudochrobactrum asaccharolyticum, a species that had never been reported for Cr(VI) removal before. Transmission electron microscopy and energy dispersive X-ray spectroscopy analysis further confirmed that strain LY6 could accumulate chromium within the cell while conducting Cr(VI) removal. The results suggested that the indigenous bacterial strain LY6 would be a new candidate for potential application in Cr(VI) pollution bioremediation.

Comparative evaluations on bio-treatment of hexavalent chromate by resting cells of Pseudochrobactrum sp. and Proteus sp. in wastewater.

Two marine bacterial strains, B5 and H24, were isolated from long-term Cr(VI) contaminated seawater and identified as Pseudochrobactrum and Proteus, respectively, based on 16S rRNA gene sequence analyses. Both strains were examined for their tolerance to Cr(VI) and other metal salts and their abilities to reduce Cr(VI) to trivalent chromium [Cr(III)]. Growing cells of Pseudochrobactrum sp. B5 and Proteus sp. H24 could tolerate Cr(VI) at a concentration of 2000 and 1500 mg/l and completely reduce 1000 mg/l Cr(VI) in LB medium within 96 and 144 h, respectively. Resting cells of the two strains were able to reduce 200mg/l Cr(VI) in Tris-HCl buffer within 16 and 24h, respectively. Furthermore, resting cells of both strains were able to reduce Cr(VI) in industrial wastewaters three times consecutively. Overall, this study provides evidence of the potential for application of chromate-reducing bacteria to direct Cr(VI) decontamination of industrial effluents.

Paenochrobactrum gallinarii gen. nov., sp. nov., isolated from air of a duck barn, and reclassification of Pseudochrobactrum glaciei as Paenochrobactrum glaciei comb. nov.

A Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (Sa25(T)) was isolated from air of a duck barn. 16S rRNA gene and recA sequence analyses clearly placed the isolate in the vicinity of the Brucella-Ochrobactrum-Pseudochrobactrum group, with the closest relative being Pseudochrobactrum glaciei KMM 3858(T). This allocation was confirmed by analyses of the quinone system (ubiquinone Q-10), fatty acid data (major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo omega8c) and polar lipid profile (major components diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and unknown aminolipid AL1; moderate amounts of three unknown polar lipids, L1-L3, an unknown aminolipid and an unknown aminophospholipid APL2). The polyamine pattern of Sa25(T) exhibited the major compound putrescine and moderate amounts of spermidine; a similar polyamine pattern with the major compound putrescine was also detected in Pseudochrobactrum glaciei KMM 3858(T). DNA-DNA hybridization of strain Sa25(T) with Pseudochrobactrum glaciei KMM 3858(T) and the type strains of the other Pseudochrobactrum species showed values ranging from 50.3 to 24.8 %, and physiological and biochemical data clearly differentiated this isolate from the described Pseudochrobactrum species. Since Sa25(T) and Pseudochrobactrum glaciei KMM 3858(T) form a distinct lineage in the 16S rRNA gene sequence-based phylogenetic tree, and this separate position is supported by unique characteristics of their polyamine patterns and polar lipid profiles, we propose the novel genus Paenochrobactrum gen. nov., with the type species Paenochrobactrum gallinarii sp. nov. (type strain Sa25(T) =CCUG 57736(T) =CCM 7656(T)) and the reclassification of Pseudochrobactrum glaciei as Paenochrobactrum glaciei comb. nov. (type strain Pi26(T) =KMM 3858(T) =NRIC 0733(T) =JCM 15115(T)).

Brevundimonas naejangsanensis sp. nov., a proteolytic bacterium isolated from soil, and reclassification of Mycoplana bullata into the genus Brevundimonas as Brevundimonas bullata comb. nov.

A Gram-negative, motile and rod-shaped bacterial strain, BIO-TAS2-2(T), of the class Alphaproteobacteria, was isolated from a soil in Korea and studied using a polyphasic taxonomic approach. Strain BIO-TAS2-2(T) grew optimally at pH 7.5-8.5 and 30 degrees C and in the presence of 0-1.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain BIO-TAS2-2(T) fell within the clade comprising species of the genus Brevundimonas, forming a coherent cluster with Brevundimonas terrae KSL-145(T) and Brevundimonas diminuta LMG 2089(T). It exhibited 16S rRNA gene sequence similarity values of 96.0-98.7 % to members of the genus Brevundimonas and Mycoplana bullata IAM 13153(T). Strain BIO-TAS2-2(T) contained Q-10 as the predominant ubiquinone and cyclo-C(18 : 1)omega7c and C(16 : 0) as the major fatty acids. The DNA G+C content was 67.0 mol%. Strain BIO-TAS2-2(T) exhibited DNA-DNA relatedness levels of 12-19 % with the type strains of phylogenetically related Brevundimonas species and M. bullata. The novel strain could be differentiated from Brevundimonas species and M. bullata by differences in phenotypic characteristics. On the basis of phenotypic, phylogenetic and genetic data, strain BIO-TAS2-2(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas naejangsanensis sp. nov. is proposed. The type strain is BIO-TAS2-2(T) (=KCTC 22631(T)=CCUG 57609(T)). In this study, it is also proposed that Mycoplana bullata be transferred to the genus Brevundimonas as Brevundimonas bullata comb. nov. (type strain TK0051(T)=ATCC 4278(T)=DSM 7126(T)=JCM 20846(T)=LMG 17157(T)).

Pseudochrobactrum lubricantis sp. nov., isolated from a metal-working fluid.

A Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (KSS 7.8(T)) was isolated from a water-mixed metal-working fluid. On the basis of 16S rRNA gene and recA sequence similarities, the isolate was clearly grouped in the genus Pseudochrobactrum. This allocation was confirmed by fatty acid data (major fatty acids: C(18 : 2)omega7c and C(19 : 0) cyclo omega8c), polar lipid profile (major components: phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine, plus moderate amounts of phosphatidylmonomethylethanolamine and unknown aminolipid AL1), quinone system (ubiquinone Q-10) and polyamine pattern (spermidine and putrescine predominant). DNA-DNA pairing with the most closely related Pseudochrobactrum species showed values ranging from 24.2 to 45.7 %, and physiological and biochemical data clearly differentiated this isolate from described Pseudochrobactrum species. This organism represents a novel species of the genus Pseudochrobactrum, for which the name Pseudochrobactrum lubricantis sp. nov. is proposed, with the type strain KSS 7.8(T) (=CCUG 56963(T)=CCM 7581(T)).

Pseudochrobactrum glaciei sp. nov., isolated from sea ice collected from Peter the Great Bay of the Sea of Japan.

An aerobic, Gram-negative, non-pigmented, non-motile bacterium, KMM 3858(T), was isolated from a sea-ice sample collected from Peter the Great Bay of the Sea of Japan, Russia, and subjected to a phenotypic and phylogenetic study. Comparative analyses based on the 16S rRNA and recA gene sequences placed strain KMM 3858(T) within the genus Pseudochrobactrum. The major chemotaxonomic characteristics were found to be the presence of phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminolipid and phosphatidylcholine, major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo, and ubiquinone Q-10, confirming the affiliation of strain KMM 3858(T) to the genus Pseudochrobactrum. On the basis of the phylogenetic analysis and the physiological and biochemical characterization, strain KMM 3858(T) should be classified as representing a novel species of the genus Pseudochrobactrum, for which the name Pseudochrobactrum glaciei sp. nov. is proposed. The type strain is strain Pi26(T) (=KMM 3858(T)=NRIC 0733(T)=JCM 15115(T)).

Daeguia caeni gen. nov., sp. nov., isolated from sludge of a textile dye works.

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, K107(T), was isolated from sludge collected from a textile dye works in Korea and its taxonomic position was investigated by means of a polyphasic analysis. Strain K107(T) contained Q-10 as the predominant ubiquinone. The major fatty acids (>10% of total fatty acids) were C(18:1)omega7c and C(18:1) 2-OH. The DNA G+C content was 57.0 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain K107(T) was closely related to the genera Mycoplana, Brucella and Ochrobactrum. Strain K107(T) exhibited 16S rRNA gene sequence similarity values of 96.3-97.1% with respect to the type strains of two Mycoplana species and 94.8-96.8% with respect to members of the genera Brucella and Ochrobactrum. A phylogenetic analysis based on recA gene sequences showed that strain K107(T) forms a distinct phylogenetic lineage within the Alphaproteobacteria. The recA gene sequence of strain K107(T) showed similarity values of 84.5% with respect to type strains of Brucella species and values of 77.6-83.1% with respect to members of the genera Pseudochrobactrum, Ochrobactrum and Mycoplana. Strain K107(T) could be differentiated from phylogenetically related genera by differences in phenotypic properties and fatty acid profiles. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain K107(T) represents a novel genus and species, for which the name Daeguia caeni gen. nov., sp. nov. is proposed. The type strain of Daeguia caeni is strain K107(T) (=KCTC 12981(T) =CCUG 54520(T)).

Brevundimonas lenta sp. nov., isolated from soil.

A Gram-negative, rod-shaped, Brevundimonas-like bacterial strain, DS-18(T), was isolated from soil in Dokdo, Korea, and its exact taxonomic position was investigated by using a polyphasic approach. Strain DS-18(T) grew optimally at pH 6.5-7.0 and 25 degrees C without NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-18(T) belonged to the genus Brevundimonas. Strain DS-18(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c and C(16 : 0) as the major fatty acids. The DNA G+C content was 68.7 mol%. Strain DS-18(T) exhibited levels of 16S rRNA gene sequence similarity of 96.3-98.7 % to the type strains of Brevundimonas species and Mycoplana bullata. Mean DNA-DNA relatedness values between strain DS-18(T) and the type strains of phylogenetically related Brevundimonas species and M. bullata were in the range 15-32 %. Strain DS-18(T) differed from Brevundimonas species and M. bullata in several phenotypic characteristics. On the basis of phenotypic, phylogenetic and genetic data, strain DS-18(T) represents a novel species of the genus Brevundimonas, for which the name Brevundimonas lenta sp. nov. is proposed. The type strain is DS-18(T) (=KCTC 12871(T) =JCM 14602(T)).

Description of Pseudochrobactrum kiredjianiae sp. nov.

A Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (strain CCUG 49584(T)), isolated from a seafood processing plant sample in New Zealand, was subjected to a polyphasic taxonomic study. On the basis of 16S rRNA and recA gene sequence similarities, the isolate was allocated to the genus Pseudochrobactrum. This was confirmed by fatty acid data (major fatty acids: C(18 : 1)omega7c and C(19 : 0) cyclo omega8c), a polar lipid profile exhibiting major characteristics of Pseudochrobactrum (phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine), quinone system Q-10 and a polyamine pattern with the predominant compounds spermidine and putrescine. DNA-DNA hybridization with the type strains of the two established species of Pseudochrobactrum and physiological and biochemical data clearly differentiated the isolate from established Pseudochrobactrum species. As a consequence, this organism represents a novel species, for which the name Pseudochrobactrum kiredjianiae sp. nov. is proposed, with the type strain CCUG 49584(T) (=CIP 109227(T)).

Adaptation of the Brucellae to their intracellular niche.

Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts.