First report of nosocomial myiasis by Lucilia cuprina (Diptera: Calliphoridae) in Costa Rica

Introducción. Las miasis hospitalarias son entidades con una importancia manifiesta en salud pública. La documentación de este tipo de casos es escasa en la literatura biomédica regional y mundial. Objetivo. Informar un caso de miasis hospitalaria en Costa Rica, donde el agente etiológico implicado fue Lucilia cuprina (Diptera: Calliphoridae). Este caso de miasis hospitalaria figura como el primer informe para Latinoamérica asociado con este agente etiológico. Presentación del caso. Una paciente de 91 años de edad, con signos de inmunosupresión, afectación grave de la función pulmonar y asistencia respiratoria mecánica, presentó larvas en ambas fosas nasales al séptimo día después del ingreso hospitalario. Varios ejemplares fueron recolectados y procesados para su identificación. La identificación taxonómica de los ejemplares recolectados estableció que la especie de los muscomorfos correspondía a L. cuprina. Conclusión. El presente constituye el primer caso de miasis hospitalaria por L. cuprina en la literatura biomédica de Costa Rica y el primero registrado en Latinoamérica.Introduction. Nosocomial myiases can be an important condition from a public health perspective. However, cases of this condition reported in regional and worldwide biomedical literature are scarce. Objective. A case of nosocomial myiasis is reported from Costa Rica, where the species involved was Lucilia cuprina (Diptera: Calliphoridae). Materials and methods. A 91-year-old patient with signs of immunosuppression, severe impairment of lung function, and mechanical ventilation presented larvae in both nostrils on the seventh day after admission. Five specimens were collected and processed for identification. Results. The taxonomic identification of the specimens established that the muscoid fly species was L. cuprina. Conclusion. This is the first case of nosocomial myiasis reported from Costa Rica and in Latin America for which the etiological agent is L. cuprina.Universidad de Costa Rica/[ED-548]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Sistema de Estudios de Posgrado::Salud::Maestría Académica en Parasitologí

The Differential Interaction of Brucella and Ochrobactrum with Innate Immunity Reveals Traits Related to the Evolution of Stealthy Pathogens

International audienceBACKGROUND: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. CONCLUSIONS/SIGNIFICANCE: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia

The differential interaction of Brucella and Ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens

Background: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. Methodology/principal Findings: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. Conclusions/significance: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia

Neutrophils are required for the control of <i>O. anthropi</i> infections.

<p>(A) Neutrophils were depleted from twenty mice by injection of the anti-neutrophil RB6 antibody and ten additional mice were instead injected with PBS alone. One group of ten mice anti-neutrophil treated and the group of ten mice injected with PBS alone were then infected intraperitoneally with 10⁹ CFU/mouse of <i>O. anthropi</i>. The last group of ten mice treated with antibody anti-neutrophil was inoculated with PBS alone. The lethality of the bacteria was recorded at the indicated times. (B) <i>O. anthropi</i> was mixed with purified rat neutrophils at a ratio of 5±4 bacteria/cell. Control bacteria were not incubated with neutrophils. At the indicated times the viable CFU were determined and the percentage of bacterial replication was calculated. In “B” Values of p<0.01 (*) and p<0.001 (**) are indicated.</p

Genomic comparison of <i>Brucella</i> 2308 and <i>O. anthropi</i> LMG3331 ORFs involved in the synthesis of surface exposed or OM PAMPs.

1<p>Although acting in the O-PS biosynthetic pathway, these proteins are involved in the steps previous to O-sugar polymerization and, therefore, the corresponding sugars belong structurally to the core oligosaccharide.</p>2<p>These bacteria differ in O-PS sugar structure and have unrelated glycosyltransferases and O-PS export systems. Accordingly, only ORFs assigned to the core and lipid A are presented in the Table. Proteins known to be involved in ancillary pathways of <i>Brucella</i> core oligosaccharide (Pgm, ManBcore and ManCcore) are highly conserved in <i>O. anthropi</i>.</p>3<p>Only ORFs attributed in the annotations in databases to the lipid A modification systems <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Raetz1" target="_blank">[83]</a> are included.</p>4<p>This ORF, although not annoted in the <i>B.abortus</i> 2308 genome, was identified by using Oant_1613 as a query in the TBLASTN program (NCBI).</p>5<p>For lipoproteins, lipoprotein annotations were obtained from both genomes and verified with the lipoP (<a href="http://www.cbs.dtu.dk/services/LipoP/" target="_blank">http://www.cbs.dtu.dk/services/LipoP/</a>) in those cases in which there were discrepancies between the two annotations.</p

<i>O. anthropi</i> infection induces an intermediate blood leukocyte response.

<p>Groups of 6 mice were inoculated intraperitoneally with10⁶ CFU/ml of the indicated bacteria, bleed at different times and leukocytes counted in blood. (A) Total leukocytes number of mice infected with the indicated bacteria. (B) Relative blood leukocyte number of mice infected with <i>B. abortus</i>. (C) Relative blood leukocyte number of mice infected with <i>S. enterica</i>. (D) Relative blood leukocyte number of mice infected with <i>O. anthropi.</i> Values of <i>O. anthropi</i> in “A” were significantly different at p<0.01 (*) and p<0.001 (**) with respect to <i>B. abortus.</i> In “B, C and D” the standard error was less that 5% at all points.</p

<i>Oa</i>LPS induces cytokine responses that lay between <i>Ba</i>LPS. and <i>Se</i>LPS.

<p>(A, B and C) Groups of 6 mice were inoculated intraperitoneally with <i>Ba</i>LPS, <i>Ec</i>LPS or <i>Oa</i>LPS at the indicated concentrations. Mice were bled at 2 or 5 h and interleukins quantitated by ELISA. (D) Bone marrow macrophages from wild type TLR2−/− and TLR4−/− knockout mice were incubated with the indicated LPSs. After incubation TNF-α was quantified in the supernatant of cells at 24 h. The insert shows Raw 264.7 macrophages incubated with 25 µg of <i>Oa</i>LPS or <i>Ba</i>LPS and at the indicated times TNF-α quantified from cell culture supernatants. Values of p<0.05 (*) and p<0.005 (**) with respect to <i>Ba</i>LPS are indicated.</p

Model of <i>Brucella, Ochrobactrum</i> and <i>Escherichia</i> lipid As and core oligosaccharide moieties. [18], [19], [45].

<p>The presence of three KDO and phosphorylated heptoses confers five negative charges to the <i>Escherichia coli</i> core oligosaccharide. The core oligosaccharide of <i>Ochrobactrum</i> LPS possesses galacturonic acid, in contrast to <i>Brucella</i> LPS core which does not possess this negatively charged acidic sugar. The differential sensitivity of <i>Escherichia</i>, <i>Ochrobactrum</i> and <i>Brucella</i> to cationic peptides expressed as sensitivity to polymyxin B (PlxB) units (U) is linked to the different LPSs properties such as uptake hydrophobic probes (NPN), PlxB binding, and LPS zeta potential (ζ) dependence after saturation with polymyxin B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Velasco2" target="_blank">[18]</a>. Notice that upon saturation with PlxB, zeta potential became positive (1 mV) for <i>Ochrobactrum</i> LPS while remaining negative (−5 mV) for <i>Brucella</i> smooth LPS, suggesting hindered access to inner targets. <i>Ochrobactrum</i> and <i>Brucella</i> LPSs does not show significant differences in lipid A structure and acyl chain fluidity and display very similar structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Velasco2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Moriyn3" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Velasco3" target="_blank">[45]</a>. It has been proposed that the quinovosamine sugar in <i>Ochrobactrum</i> and <i>Brucella</i> LPS links the O core oligosaccharide with the O-chain polysaccharide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Gonzlez1" target="_blank">[21]</a>.</p

<i>O. anthropi</i> induces TNF-α that lays between <i>B. abortus</i> and <i>S. enterica</i>.

<p>(A) Murine Raw 264.7 macrophages were infected for 30 min with <i>O. anthropi</i> or <i>B. abortus</i> at a MOI of 500. Extracellular bacteria were killed by addition of gentamicin. At the indicated times samples from the culture media were taken and processed for TNF-α quantification. (B) Groups of 6 mice were inoculated intraperitoneally with <i>O. anthropi</i>. At the indicated times mice were bled and TNF-α levels were determined. The levels of TNF-α induction after 3 h of an equivalent inoculation of <i>B. abortus</i> or <i>S. enterica</i>. Value of p<0.001 (**) is indicated.</p