\u3cem\u3eRickettsia felis\u3c/em\u3e in Cat Fleas, \u3cem\u3eCtenocephalides felis\u3c/em\u3e Parasitizing Opossums, San Bernardino County, California

Los Angeles and Orange Counties are known endemic areas for murine typhus in California; however, no recent reports of flea-borne rickettsioses are known from adjacent San Bernardino County. Sixty-five opossums (Didelphis virginiana) were trapped in the suburban residential and industrial zones of the southwestern part of San Bernardino County in 2007. Sixty out of 65 opossums were infested with fleas, primarily cat fleas, Ctenocephalides felis (Bouché, 1835). The flea minimum infection rate with Rickettsia felis was 13.3% in pooled samples and the prevalence was 23.7% in single fleas, with two gltA genotypes detected. In spite of historic records of murine typhus in this area, no evidence for circulation of R. typhi in fleas was found during the present study. Factors contributing to the absence of R. typhi in these cat fleas in contrast to its presence in cat fleas from Orange and Los Angeles Counties are unknown and need to be investigated further in San Bernardino County

Flow cytofluorimetric detection and immunophenotyping of platelet-monocyte complexes in peripheral blood

Activated platelets aggregate with monocytes by binding membrane bound molecules. Platelet-monocyte interaction is considered to underlie pathophysiological mechanisms bridging thrombosis and inflammation. Detection and analysis of platelet-monocyte complexes (PMC) provide means for revealing their physiological and pathogenetic roles and are instrumental in the diagnostics of various pathological conditions including obstetric complications. The aim of the study was to develop the method of quantitative determination of peripheral blood PMC, that preserve phenotypic features of platelets and monocytes, and to reveal their changes by ex vivo analysis. The suggested procedure includes immediate fixation of blood sample, immunocytochemical staining with fluorochrome-conjugated specific antibodies against markers of activation and differentiation followed by lysis of erythrocytes, and flow cytometric analysis. Fourteen samples of peripheral blood from patients with history of pregnancy complication were obtained in first trimester of ongoing pregnancy and analyzed. It was demonstrated that quantitative and qualitative in vivo characteristics of PMC remained unchanged in fixed samples, whereas the number of PMC and expression levels of the markers of platelet and monocyte activation dramatically increased in the unfixed blood. The set of monoclonal antibodies and gating strategies, used in this study, ensure phenotyping and evaluation of percentage/absolute count of PMC in the total monocyte population (CD45+CD14+) and in the subpopulations of classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14lowCD16+) monocytes. This approach provides insight into the participation of different monocyte subsets in the formation of PMC and their roles in physiological and pathophysiological processes. In some samples, elevated PMC proportion was observed, accompanied by significant increase in the expression of platelet activation marker CD62P and decrease in the expression of its monocytic ligand CD162. These changes suggested altered activation of PMC and their participation in the pathophysiological mechanisms of some pregnancy complications. Immunophenotyping of PMC affords an opportunity to characterize their proinflammatory, procoagulant and adhesive properties; these results can be used for research and diagnostics. In particular, the method is suitable for detection and phenotyping of PMC in pregnancy complications and other pathological conditions associated with the disorders of hemostasis and thrombosis

Rickettsia parkeri in Amblyomma americanum Ticks, Tennessee and Georgia, USA

To determine the geographic distribution of the newly recognized human pathogen Rickettsia parkeri, we looked for this organism in ticks from Tennessee and Georgia, USA. Using PCR and sequence analysis, we identified R. parkeri in 2 Amblyomma americanum ticks. This rickettsiosis may be underdiagnosed in the eastern United States

Effect of particle properties of powders on the generation and transmission of raman scattering

Transmission Raman measurements of a 1 mm thick sulfur-containing disk were made at different positions as it was moved through 4 mm of aspirin (150-212 mu m) or microcrystalline cellulose (Avicel) of different size ranges (<38, 53-106, and 150-212 mu m). The transmission Raman intensity of the sulfur interlayer at 218 cm(-1) was lower when the disk was placed at the top or bottom of the powder bed, compared to positions within the bed and the difference between the sulfur intensity at the outer and inner positions increased with Avicel particle size. Also, the positional intensity difference was smaller for needle-shaped aspirin than for granular Avicel of the same size. The attenuation coefficients for the propagation of the exciting laser and transmitted Raman photons through the individual powders were the same but decreased as the particle size of Avicel increased; also, the attenuation coefficients for propagation through 150-212 mu m aspirin were almost half of those through similar sized Avicel particles. The study has demonstrated that particulate size and type affect transmitted Raman intensities and, consequently, such factors need to be considered in the analysis of powders, especially if particle properties vary between the samples

Stellar structure and compact objects before 1940: Towards relativistic astrophysics

Since the mid-1920s, different strands of research used stars as "physics laboratories" for investigating the nature of matter under extreme densities and pressures, impossible to realize on Earth. To trace this process this paper is following the evolution of the concept of a dense core in stars, which was important both for an understanding of stellar evolution and as a testing ground for the fast-evolving field of nuclear physics. In spite of the divide between physicists and astrophysicists, some key actors working in the cross-fertilized soil of overlapping but different scientific cultures formulated models and tentative theories that gradually evolved into more realistic and structured astrophysical objects. These investigations culminated in the first contact with general relativity in 1939, when J. Robert Oppenheimer and his students George Volkoff and Hartland Snyder systematically applied the theory to the dense core of a collapsing neutron star. This pioneering application of Einstein's theory to an astrophysical compact object can be regarded as a milestone in the path eventually leading to the emergence of relativistic astrophysics in the early 1960s.Comment: 83 pages, 4 figures, submitted to the European Physical Journal

The mechanisms of antibiotic resistance in major pathogens of purulent-inflammatory complications in cancer patients

The problem of microbial antibiotic resistance and investigation of its underlying mechanisms is of paramount importance for all fields of clinical medicine, including oncology. The aim of the study was to examine the mechanisms of antibiotic resistance for major pathogens causing purulent-inflammatory complications in cancer patients. Materials and methods. In 2012—2015 there was conducted a prospective examination of 184 cancer patients, including 67 patients at the Department of Surgery no. 1 and 117 patients at the Intensive Care Unit of the Krasnoyarsk Regional Clinical Oncology Center named after A.I. Kryzhanovsky. For this, we collected bronchoalveolar lavage fluid, wound discharge and investigated the material by using bacteriological method, as well as MALDI-TOF. Antibiotic sensitivity was studied as follows: disco-diffusion, double disc method, carbapenem inactivation method, staphylococcal sensitivity — by screening method, PCR, E-test method, and serial dilutions in Muller-Hinton broth. Genotyping and antibiotic resistance mechanisms study were performed by using PCR, M-PCR, and sequencing. The WHONET program (WHO) was used, with significance level set at p &lt; 0.05. Results. Microbiological examination of bronchoalveolar lavage fluid and wound discharge samples allowed to uncover prevalent associations of multi-resistant (MDR) and extremely resistant pathogens (XDR). The microflora of the lower respiratory tract and in the wound secretion in cancer patients were found to be dominated by non-fermenting Gram-negative bacteria reaching up to 44.5% and 48%, respectively; as well as order Enterobacteriales found in 24% and 34.9%, respectively; Gram-positive bacteria — 24% and 17.1%, respectively. Imipenem- and/or meropenem-resistant P. aeruginosa and A. baumannii, K. pneumoniae strains, were assessed for MBL production phenotypically, as well as the genes of the most common VIM, IMP types, whereas A. baumannii — for OXA-23, OXA-40, and OXA-58; and K. pneumoniae — for OXA-48. 20 strains and 16 strains of P. aeruginosa and A. baumannii, respectively, were studied by PCR. It was found that A. baumannii strains formed no MBL, but 56.3% of A. baumannii isolates (9 strains) produced OXA-23 and OXA-40 carbapenemases. Among P. aeruginosa strains there were three of them which possessed VIM (15.0%), whereas the remaining strains formed no MBL, but were resistant to carbapenems being associated with other resistance mechanisms, e.g. efflux, decreased permeability of cell wall etc. Among 6 isolates of K. pneumoniae, 1 strain produced OXA-48. In cancer patients, the percentage of methicillin-resistant strains among all members of the genus Staphylococcus was 48.9% (4 strains belonged to MRSA). PVL- MRSA strains belonged to the clones ST239/spa3(t037)/SCCmecIIIA/tst,sek,seq+ (75%) and ST8/ spa1(t008)/SCCmecIVc/sea+ (25%). MRSA ST239 showed multiple antibiotic resistance: to aminoglycosides (aacA-aphD, aadD genes were detected), linkcosamides/macrolides (the ermA gene was detected), fluoroquinolones (mutations in the GyrA gene — Ser84Leu; in GrlA- Ser80Phe), rifampicin (MIC more than 128 gg/ml; mutations in the rpoB gene are His481Asn, Ile527Met), sulfamethoxazole, tetracycline (tetM gene), and chloramphenicol (66.7% of isolates, the cat gene encoding chloramphenicol acetyl transferase was detected); but sensitive to vancomycin (MIC 1.0 gg/ ml), linezolid in 100% of cases. MRSA ST8 are resistant to aminoglycosides (aacA-aphD, aadD genes), lincosamides/macrolides (ermC gene), tetracyclines (tetK gene), chloramphenicol (cat gene); and 100% sensitive to fluoroquinolones, rifampicin (MIC 0.006 gg/ml), sulfamethaxazole, vancomycin (MIC 1.0 gg/ml), daptomycin (MIC 0.094 gg/ml), linezolid (MIC 0.75 gg/ml). Conclusion. Thus, it was found that members of the order Enterobacteriales, A. baumannii, P. aeruginosa and MRSA retain high resistance to a large number of antibacterial drugs of almost all classes. These data should be taken into account while choosing proper antibiotic therapy, as well as controlling spread of nosocomial infections caused by multiresistant microorganisms

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

BACKGROUND: Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular alpha-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs). METHODOLOGY/PRINCIPAL FINDINGS: We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (approximately 1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions. CONCLUSION/SIGNIFICANCE: Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets

Parallel Evolution of a Type IV Secretion System in Radiating Lineages of the Host-Restricted Bacterial Pathogen Bartonella

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree