Reclassification of Ochrobactrum lupini as a later heterotypic synonym of Ochrobactrum anthropi based on whole-genome sequence analysis.

The genus Ochrobactrum belongs to the family Brucellaceae and its members are known to be adapted to a wide range of ecological niches. Ochrobactrum anthropi ATCC 49188T and Ochrobactrum lupini LUP21T are strains isolated from human clinical and plant root nodule samples, respectively, which share high similarity for phylogenetic markers (i.e 100 % for 16S rRNA, 99.9 % for dnaK and 99.35 % for rpoB). In this work, multiple genome average nucleotide identity (ANI) approaches, digital DNA-DNA hybridization (dDDH) and phylogenetic analysis were performed in order to investigate the taxonomic relationship between O. anthropi ATCC 49188T, O. lupini LUP21T, and other five type strains from the genus Ochrobactrum. Whole-genome comparisons demonstrated that O. lupini LUP21T and the Ochrobactrum genus type species, O. anthropi ATCC 49188T, share 97.55 % of ANIb, 98.25 % of ANIm, 97.99 % of gANI, 97.94 % of OrthoANI and 83.9 % of dDDH, which exceed the species delineation thresholds. These strains are also closely related in phylogenies reconstructed from a concatenation of 1193 sequences from single-copy ortholog genes. A review of their profiles revealed that O. anthropi ATCC 49188T and O. lupini LUP21T do not present pronounced differences at phenotypic and chemotaxonomic levels. Considering phylogenetic, genomic, phenotypic and chemotaxonomic data, O. lupini should be considered a later heterotypic synonym of O. anthropi.

Brucella suis bacteremia misidentified as Ochrobactrum anthropi by the VITEK 2 system.

Ochrobactrum and Brucella are genetically related genera of the family Brucellaceae, sharing 98.8% rRNA similarity. Because of their phenotypic similarity, Ochrobactrum can be miscoded as Brucella by automated identification systems. The misidentification on blood cultures (BCs) of B. suis as O. anthropi by the VITEK 2 system is herein described. A 67-year-old male with a prosthetic mitral valve and fever was admitted with bacteremia due to a Gram-negative coccobacillus identified as O. anthropi by VITEK 2. The patient's fever persisted along with positive blood cultures despite specific antimicrobial treatment. Due to this adverse outcome, the patient was interrogated again and admitted having domestic swine. Serological tests were positive for acute brucellosis. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of BC strains identified B. suis biovar 1. Timely identification of Brucella is essential for providing proper treatment to the patient and for advising safe handling of laboratory cultures in biological safety cabinets to prevent laboratory-acquired infection. Countries where brucellosis is endemic must be aware of this possibility.

Ochrobactrum anthropi bacteremia in a preterm infant with cystic fibrosis.

Ochrobactrum anthropi infection in newborn patients is rare, and the treatment is challenging because of its widespread and unpredictable resistance to antimicrobial agents and discrepancies between in vitro susceptibility and in vivo efficacy. We report the clinical and microbiological characteristics of Ochrobactrum anthropi bacteremia in a preterm patient.

Colonization by endophytic Ochrobactrum anthropi†Mn1 promotes growth of Jerusalem artichoke.

The Ochrobactrum anthropi Mn1 strain, taxonomically identified using 16S ribosomal DNA sequence, was isolated from roots of Jerusalem artichoke. Its endophytic colonization was investigated microscopically using green fluorescent protein introduced by vector pHC60. The strain entered Jerusalem artichoke tissues through the root, and was localized in the roots and stems. The plant growth-promoting (PGP) effects of O. anthropi Mn1 were assessed in greenhouse as well as field trials with different nitrogen supplies. Only under moderate to ample nitrogen supply, could O. anthropi Mn1 promoted growth of host plant. The PGP effects of the strain were symbiotic nitrogen fixation, root morphological optimization and enhanced nutrient uptake. We hypothesize that the symbiotic interspecies interaction might be quorum sensing related.

Ochrobactrum anthropi bacteremia in a preterm infant with cystic fibrosis

Ochrobactrum anthropi infection in newborn patients is rare, and the treatment is challenging because of its widespread and unpredictable resistance to antimicrobial agents and discrepancies between in vitro susceptibility and in vivo efficacy. We report the clinical and microbiological characteristics of Ochrobactrum anthropi bacteremia in a preterm patient.

Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

BACKGROUND: Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram. RESULTS: Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates. CONCLUSIONS: Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

Clinical characteristics of Ochrobactrum anthropi bacteremia.

The clinical picture of Ochrobactrum anthropi infection is not well described because the infection is rare in humans and identification of the pathogen is difficult. We present a case of O. anthropi bacteremia that was initially misidentified as Ralstonia paucula and later identified by 16S rRNA sequencing and recA analysis.

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp.

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C-P lyase incapable of degrading GP (C-P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C-P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C-P lyase II. O. anthropi GPK 3 also degraded MP via C-P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.

Genome of Ochrobactrum anthropi ATCC 49188 T, a versatile opportunistic pathogen and symbiont of several eukaryotic hosts.

Ochrobactrum anthropi is a common soil alphaproteobacterium that colonizes a wide spectrum of organisms and is being increasingly recognized as an opportunistic human pathogen. Potentially life-threatening infections, such as endocarditis, are included in the list of reported O. anthropi infections. These reports, together with the scant number of studies and the organism's phylogenetic proximity to the highly pathogenic brucellae, make O. anthropi an attractive model of bacterial pathogenicity. Here we report the genome sequence of the type strain O. anthropi ATCC 49188, which revealed the presence of two chromosomes and four plasmids.

Ribosomal RNA sequence analysis of Brucella infection misidentified as Ochrobactrum anthropi infection.

A Brucella isolate was identified from purulent material collected during a hip surgery. Two previous blood cultures from the same patient yielded Ochrobactrum anthropi. After rRNA sequencing, all the isolates were identified as Brucella species and subsequently serotyped as Brucella suis. Misidentification of Brucella species remains a problem with bacterial identification systems.

Multilocus sequence typing supports the hypothesis that Ochrobactrum anthropi displays a human-associated subpopulation.

BACKGROUND: Ochrobactrum anthropi is a versatile bacterial species with strains living in very diverse habitats. It is increasingly recognized as opportunistic pathogen in hospitalized patients. The population biology of the species particularly with regard to the characteristics of the human isolates is being investigated. To address this issue, we proposed a polyphasic approach consisting in Multi-Locus Sequence Typing (MLST), multi-locus phylogeny, genomic-based fingerprinting by pulsed-field gel electrophoresis (PFGE) and antibiotyping. RESULTS: We tested a population of 70 O. anthropi clinical (n = 43) and environmental (n = 24) isolates as well as the type strain O. anthropi ATCC49188T and 2 strains of Ochrobactrum lupini and Ochrobactrum cytisi isolated from plant nodules. A Multi-Locus Sequence Typing (MLST) scheme for O. anthropi is proposed here for the first time. It was based on 7 genes (3490 nucleotides) evolving mostly by neutral mutations. The MLST approach suggested an epidemic population structure. A major clonal complex corresponded to a human-associated lineage since it exclusively contained clinical isolates. Genomic fingerprinting separated isolates displaying the same sequence type but it did not detect a population structure that could be related to the origin of the strains. None of the molecular method allowed the definition of particular lineages associated to the host-bacteria relationship (carriage, colonisation or infection). Antibiotyping was the least discriminative method. CONCLUSION: The results reveal a human-associated subpopulation in our collection of strains. The emergence of this clonal complex was probably not driven by the antibiotic selective pressure. Therefore, we hypothesise that the versatile species O. anthropi could be considered as a human-specialized opportunistic pathogen.

Novel denitrifying bacterium Ochrobactrum anthropi YD50.2 tolerates high levels of reactive nitrogen oxides.

Most studies of bacterial denitrification have used nitrate (NO3-) as the first electron acceptor, whereas relatively less is understood about nitrite (NO2-) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO2- (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the alpha subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO2-, as well as NO3-. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO2- reductase containing copper that was involved in bacterial NO2- reduction. The strain denitrified up to 40 mM NO2- to dinitrogen under anaerobic conditions in which other denitrifiers or NO3- reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO2-. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188(T) proliferated even in the presence of higher levels of NO2- (100 mM), and both were considerably more resistant to acidic NO2- than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.

Isolation of Brucella microti from mandibular lymph nodes of red foxes, Vulpes vulpes, in lower Austria.

From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.

Ochrobactrum anthropi bacteremia in a preterm infant with meconium peritonitis.

Ochrobactrum anthropi is a non-fermenting gram-negative rod that was identified as a pathogenic microorganism during the past decade. O. anthropi is extensively distributed in the environment, and has been found in hospital and environmental water sources. O. anthropi infection is rare in childhood. We report a case of O. anthropi bacteremia in a preterm infant with a peritoneal lavage catheter and meconium peritonitis.

Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis.

A recA-PCR restriction fragment length polymorphism assay was developed to study intraspecies variation among Ochrobactrum anthropi. Primers deduced from the known recA gene sequence of the genetically closely related genus Brucella allowed the specific amplification of a 1065 bp recA fragment from each of the 38 O. anthropi and the eight Brucella strains investigated. RecA was also amplified from the type strains of O. intermedium, O. tritici, and O. lupini but could not be generated from O. grignonense and O. gallinifaecis. Subsequent comparative recA sequence- and HaeIII-recA restriction fragment length polymorphism analysis identified nine different genospecies among the tested 38 O. anthropi isolates, whereas the recA sequences of the Brucella spp. were indistinguishable. Furthermore, Brucella spp., O. anthropi, O. intermedium, and O. tritici were clearly separated from each other by means of their recA sequences and HaeIII restriction patterns. Five strains of uncertain species status listed in the Culture Collection University of Göteborg bacterial culture collection as O. anthropi were characterized by recA analysis, and their phylogenetic position within the Brucella-Ochrobactrum group was determined. In summary, recA-sequence analysis provides a new reliable molecular subtyping tool to study the phylogeny of the Ochrobactrum taxon at both the inter- and intraspecies level.

D,L-Hydantoinase activity of an Ochrobactrum anthropi strain.

AIMS: A microorganism with the ability to release methionine from D,L-(2-methylthioethyl) hydantoin (strain 245) was isolated from soil. The aim of this study was the identification of the strain and the adjustment of the conditions of growth and of the enzymatic reaction, in order to achieve high specific activities of bioconversion of the hydantoin. METHODS AND RESULTS: Strain 245 was identified as Ochrobactrum anthropi. The strain grew at alkaline pH (up to 10.0) and its hydantoinase activity was found to be inducible by the substrate D,L-(2-methylthioethyl) hydantoin. The enzyme is also alkalostable, with a pH optimum of 9.0. Under these conditions, hydantoinase activity was significantly enhanced and its half life prolonged when 200 mmol l-1 ammonium and phosphate were added. The addition of Ca2+, Na+, Cu2+, Co2+, Mg2+, Zn2+ or Fe3+ (0.5 mmol l-1) to the reaction mixture increased the hydantoinase activity of strain 245 up to tenfold after 24 h of incubation, compared with unamended controls. CONCLUSION: The adequate adjustment of some environmental parameters (pH, addition of inducer, presence of ammonium, phosphate, heavy metals and other ions) can considerably increase the D, L-hydantoinase activity of strain 245. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here set up the initial conditions for a further application of strain 245 in the production of methionine from hydantoine.

Characterization, cloning and sequence analysis of the inducible Ochrobactrum anthropi AmpC beta-lactamase.

Ochrobactrum anthropi is resistant to most cephalosporins and penicillins due, at least in part, to the inducible expression of a single beta-lactamase. The beta-lactamase gene has been cloned and sequenced. It encodes an AmpC-type class 1 serine active-site enzyme that hydrolyses mainly cephalosporins and is resistant to inhibition by clavulanic acid. Expression of the ampC gene is inducible via a typical AmpR regulator, which is encoded upstream of ampC. Inducible expression is retained following cloning of O. anthropi ampR-ampC into Escherichia coli, confirming that the signal for AmpR activation in O. anthropi is the same as that used in the Enterobacteriaceae. This is the first reported example of an AmpC beta-lactamase outside of the gamma-subdivision of the bacterial kingdom. Genomic searches of other non-gamma-subdivision bacteria revealed a homologous ampR-ampC cluster in the plant symbiont, Sinorhizobium meliloti.

Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida).

Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance.