Development of a system for genetic manipulation of Bartonella bacilliformis.

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.     

A phage in Bartonella bacilliformis.

Bacteriophage-like particles were found in Bartonella bacilliformis culture. The particles consisted of head (icosahedral), 40 nm in diameter, and tail, 16 nm in length.     

Hyper-reactivity of rabbits sensitized with Bartonella bacilliformis

Mitchell, Paul D. (West Virginia University Medical Center, Morgantown), and John M. Slack. Hyper-reactivity of rabbits sensitized with Bartonella bacilliformis. J. Bacteriol. 92:769-779. 1966.-Sensitization with viable cells of Bartonella bacilliformis increased the susceptibility of rabbits to the lethality of subsequently administered Bartonella metabolites. In animals sensitized with 3 weekly doses of the organism, this susceptible state of hyper-reactivity was maximal between 4 and 14 days postsensitization (primary hyper-reactive state) and persisted for at least 4 weeks, after which the animals were nonreactive (tolerant state). However, on the 84th day, the susceptible state could once again be demonstrated (secondary hyper-reactive state). Animals sensitized with only 1 or 2 weekly doses of the organism were rendered nearly as susceptible, but the time interval between the primary and secondary states of hyper-reactivity was much shorter, indicating that the hyper-reactive states were dependent upon the degree of sensitization. Symptoms displayed by such animals demonstrated an association with endotoxic shock and an anaphylactic or immediate hypersensitive response, the reaction frequently being severe enough to lead to the death of the animal within 24 hr. The histological findings were those of the generalized Shwartzman phenomenon with indications of shock. Such hyper-reactive animals produced an early-occurring, precipitating antibody specific for the somatic, endotoxic component of various gram-positive microorganisms. Injection of sera from the hyper-reactive animals into normal, nonsensitized animals resulted in a passive, hyper-reactive state in the latter animals. A distinct relationship between the levels of specific antibody and the degree of demonstrable hyper-reactivity was observed. This relationship is discussed relative to the histological findings of the hyper-reactive animals.     

Physical map of the Bartonella bacilliformis genome.

The genome of Bartonella bacilliformis was shown to be a single circular DNA molecule of about 1,600 kbp having six NotI, four SfiI, and two CeuI sites. A physical map of the DNA was constructed by contour-clamped homogeneous electric field pulsed-field gel electrophoresis of DNA restriction fragments. rRNA operons, the invasion-associated locus, and a flagellin gene were located on the map by hybridization.     

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples

BackgroundThe lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches.Conclusions/SignificanceFrom the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.     

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.     

实时荧光定量TaqMan-MGB探针法检测杆菌样巴尔通体

【目的】应用Taq Man探针实时荧光定量PCR技术建立特异性强、敏感性高和稳定性好的快速杆菌样巴尔通体检测方法。【方法】应用生物信息学方法查找杆菌样巴尔通体特有基因,从中筛选出一段特有的基因序列为模板设计探针和引物。通过比较Ct值和荧光强度确定扩增反应的最佳退火温度、探针和引物浓度;将扩增产物连接到p EASY-T载体上制备标准品,绘制标准曲线,分析扩增效率和线性关系;评估方法的特异性、敏感性及重复性。【结果】优化后退火温度为60°C,探针和引物浓度均为200 nmol/L,反应体系20μL。特异性实验显示只有杆菌样巴尔通体扩增出荧光信号,其他种属细菌均未见荧光信号;标准曲线线性关系良好(R2=1),扩增效率E=98.18%;最低检出限为每个PCR反应3个拷贝;组内和组间的变异系数CV值分别为0.21%–0.42%和0.29%–0.59%,在允许范围内。【结论】研究建立的实时荧光定量Taq Man-MGB探针法特异性强、灵敏度高、稳定性好,可快速检测鉴定杆菌样巴尔通体,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

An Outbreak of Bartonella bacilliformis in an Endemic Andean Community

Background

Bartonellosis affects small Andean communities in Peru, Colombia and Ecuador. Research in this area has been limited.

Methods

Retrospective review of 191 cases of bartonellosis managed in Caraz District Hospital, Peru, during the last outbreak (2003).

Results

The majority of cases (65%) were 14 years old and younger. There was a peak in acute cases after the rainy season; chronic cases presented more constantly throughout the year. The sensitivity of blood smear against blood culture in acute disease was 25%. The most commonly used treatment for chronic disease was rifampicin; chloramphenicol was used to treat most acute cases. Complications arose in 6.8% and there were no deaths.

Conclusions

Diagnostic and treatment algorithms for acute and chronic bartonellosis have been developed without a strong evidence base. Preparation of ready-to-go operational research protocols for future outbreaks would strengthen the evidence base for diagnostic and treatment strategies and enhance opportunities for control.     

Taxonomic considerations of Bartonella bacilliformis based on phylogenetic and phenotypic characteristics

The 16S-rRNA gene of Bartonella bacilliformis was amplified using the polymerase-chain reaction (PCR). The amplification product was sequenced using a linear-PCR procedure and compared with other published 16S-rRNA sequences. The results of this analysis placed B. bacilliformis in the alpha subgroup of the proteobacteria, and more specifically demonstrated its close phylogenetic relationship to Rochalimaea quintana. This relationship is supported by similarities in the size and mean base composition of the genomes of the two species, and by shared phenotypic characteristics.     

趋磁螺菌遗传操作体系的建立及磁小体缺失突变株的筛选

由于MagnetospirillumgryphiswaldenseMSR 1缺少简便有效的遗传操作体系和对常见抗生素的抗性 ,致使对该菌磁小体生物合成的机制等研究工作进展缓慢。为此建立了一套比较简便有效的遗传操作体系 ,其中包括 :以平板封膜培养技术获得单菌落、在选择性培养液中进行接合转移遗传因子 ,以液体培养和磁铁吸附技术筛选突变子。利用此体系 ,通过接合转座诱变技术 ,获得了 2个磁小体缺失突变株 ,为研究该菌磁小体合成的分子遗传学提供了技术支撑     

Susceptibility of Owl Monkeys (Aotus nancymaae) to Experimental Infection with Bartonella bacilliformis

Bartonellosis, caused by Bartonella bacilliformis, is a clinically significant disease in parts of South America, where it is characterized by fever and hemolytic anemia during the often-fatal acute stage and warty skin eruptions during chronic disease. In this study, we evaluated owl monkeys (Aotus nancymaae) as a potential model for studying the immunogenicity and pathology of bartonellosis. Two groups of animals (n = 3 per group) received either 9.5 × 10⁷ CFU B. bacilliformis by the ID route or 1.1 × 10⁶ CFU by the IV route and were followed for 140 d. Animals were evaluated by physical exam, complete blood count or hematocrit (or both); infection was confirmed by Giemsa staining of blood smears, PCR amplification, and blood culture. On days 7 and 21, Giemsa-stained blood smears from both groups contained organisms (1% to 4% of erythrocytes). All blood cultures and PCR tests were negative. Complete blood counts and chemistry panels showed no difference from baseline. Serology revealed a greater than 4-fold increase in the IgM titer (compared with baseline levels) in the 3 animals from the ID group and 1 animal from the IV group. On day 35, a dermal lesion was excised from the inguinal region of 1 monkey from each group, with a second lesion excised on day 84 from the same monkey in the IV group. However the histopathology and immunostaining of these samples were not consistent with B. bacilliformis. The present study shows that owl monkeys can be infected with B. bacilliformis, but additional dosage studies are necessary to evaluate the usefulness of this species as a disease model for human bartonellosis.Abbreviations: CBC, complete blood countBartonellosis (Carrion disease, Oroya fever, verruga peruana) is a complicated, multistage infectious disease caused by the bacterium Bartonella bacilliformis. B. bacilliformis-associated disease is limited almost exclusively to the Andes mountain region of South America because of the limited habitat of its sand fly vector, Lutzomia verrucarum. In the valleys of the Andes, approximately 60% of the human population is seropositive for the bacterium, and 5% to 10% of the population are active carriers of the disease.⁹ For 1997 through 2005, the Peruvian Ministry of Health reported a 10-fold increase in the incidence of bartonellosis (948 to 10,390 cases).¹⁶ Oroya fever is the hemolytic, immunosuppressive manifestation of acute infection¹ and has a case fatality rate of as high as 90% if left untreated; death is often associated with bacterial and protozoal superinfections.^2,20 The chronic stage of bartonellosis produces disfiguring warty, vascular nodules on the skin and is termed verruga peruana (Peruvian wart disease), which has a prolonged course but ultimately resolves and seldom results in death.⁴ To date, the organism has failed to be isolated from an animal reservoir, suggesting that eradication of the disease could be achieved by successful vaccination of the human population where the disease is endemic.Rhesus macaques can be infected with B. bacilliformis and develop Oroya fever (without the severe anemia) and granulomatous nodules resembling verruga peruana.¹⁴ Later experiments with mice, hamsters, guinea pigs, rabbits, and rhesus monkeys revealed their susceptibility to infection and found that the rabbit was the most susceptible.¹⁹ In these previous studies, however, the animals were inoculated by direct injection with biologic samples from infected patients, and no dose quantitation was performed. The purpose of the present study was to inoculate owl monkeys (Aotus nancymaae) with a defined inoculum of B. bacilliformis to determine their susceptibility to infection. We selected owl monkeys because they are native to Peru and are representative of New World nonhuman primates; previous studies with Old World primates produced conflicting results.^14,19 Small size, ease of handling, and lack of Cercopithicine herpesvirus 1 make owl monkeys an ideal nonhuman primate to use as an animal model. With the establishment of a nonhuman primate model that approximates the disease in humans, new strategies for the prevention and treatment of bartonellosis could be developed.     

Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria

Bacillary angiomatosis (BA) and chronic bartonellosis are bacterial infections of humans which result in an unusual vascular proliferative tissue response. In order to determine their phylogenetic relationships, we have determined greater than 95% of the 16S rRNA sequences for these two organisms by amplification directly from infected BA tissue and from a Bartonella bacilliformis lyophilized culture. The BA agent and B. bacilliformis are closely related alpha-proteobacteria (98.5%), although the BA agent is more closely related to Rochalimaea quintana (99.1%). Contrary to previous belief, the BA agent is distinct from, and less closely related to, the cat scratch bacillus (Afipia felis) (90.7%). We propose a novel secondary structure in a hypervariable region of the 16S rRNA which is useful for alignment of primary sequences and which may be useful for design of nucleic acid probes.     

The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ.

A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis.     

Multi-locus sequence analysis reveals profound genetic diversity among isolates of the human pathogen Bartonella bacilliformis

Bartonella bacilliformis is the aetiological agent of human bartonellosis, a potentially life threatening infection of significant public health concern in the Andean region of South America. Human bartonellosis has long been recognised in the region but a recent upsurge in the number of cases of the disease and an apparent expansion of its geographical distribution have re-emphasized its contemporary medical importance. Here, we describe the development of a multi-locus sequence typing (MLST) scheme for B. bacilliformis and its application to an archive of 43 isolates collected from patients across Peru. MLST identified eight sequence types among these isolates and the delineation of these was generally congruent with those of the previously described typing scheme. Phylogenetic analysis based on concatenated sequence data derived from MLST loci revealed that seven of the eight sequence types were closely related to one another; however, one sequence type, ST8, exhibited profound evolutionary divergence from the others. The extent of this divergence was akin to that observed between other members of the Bartonella genus, suggesting that ST8 strains may be better considered as members of a novel Bartonella genospecies.