Dissemination of blaIMP-8 among Enterobacteriaceae isolates from a Buenos Aires Hospital

Entre agosto de 2008 y diciembre de 2011 se detectaron en el Hospital Interzonal General de Agudos «Evita» de Lanús 6 aislamientos de enterobacterias productoras de metalo- β-lactamasas, distribuidos en tres especies: Enterobacter cloacae (4), Klebsiella oxytoca (1) y Citrobacter freundii (1). Los seis aislamientos presentaron un perfi l de multirresistencia y se confi rmó la presencia del gen blaIMP-8. Cinco aislamientos además expresaron la β-lactamasa de espectro extendido PER-2. El gen blaIMP-8 fue hallado como primer casete de un integrón de clase 1. Sin embargo, la secuencia 3´ conservada no pudo detectarse en tres aislamientos. En todos los casos, el gen blaIMP-8 fue transferido por conjugación a Escherichia coli resistente a azida. Mediante PFGE se observó que los cuatro aislamientos de E. cloacae no estuvieron genéticamente relacionados. Estos son los primeros hallazgos de metalo-β-lactamasas en la institución, que sugieren una posible diseminación horizontal del gen blaIMP-8 intra e interespecies.From August 2008 to December 2011, six metallo-β-lactamase-producing isolates, four Enterobacter cloacae, one Klebsiella oxytoca and one Citrobacter freundii, were detected at Hospital Interzonal General de Agudos “Evita” in Lanús. All six isolates showed multiresistant profi les and the presence of the blaIMP-8 gene. Five isolates also expressed PER-2 extended spectrum β-lactamase. The blaIMP-8 gene was found as the fi rst cassette in a class 1 integron. However, the 3´ conserved sequence could not be detected in three isolates. In all cases, blaIMP-8 was transferred by conjugation to azide-resistant Escherichia coli J53. PFGE analysis revealed that the four E. cloacae isolates were not genetically related. These are the fi rst metallo-β-lactamases detected in this institution and our results suggest a possible intra- and inter-species horizontal dissemination of blaIMP-8.Fil: Togneri, Ana M.. Hospital Interzonal General de Agudos Evita. Laboratorio de Bacteriología; ArgentinaFil: Gómez, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Area de Antimicrobianos; ArgentinaFil: Podestá, Laura B.. Hospital Interzonal General de Agudos Evita. Laboratorio de Bacteriología; ArgentinaFil: Pérez, Marcela P.. Hospital Interzonal General de Agudos Evita. Laboratorio de Bacteriología; ArgentinaFil: Faccone, Diego Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Area de Antimicrobianos; ArgentinaFil: Ríos, Lidia E.. Hospital Interzonal General de Agudos Evita. Laboratorio de Bacteriología; ArgentinaFil: Gañete, Marcelo A.. Hospital Interzonal General de Agudos Evita. Laboratorio de Bacteriología; ArgentinaFil: Anchordoqui, María S. . Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Area de Antimicrobianos; ArgentinaFil: Pasterán, Fernando G.. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Area de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Corso, Alejandra C.. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Area de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Low molecular weight hyaluronan-pulsed human dendritic cells showed increased migration capacity and induced resistance to tumor chemoattraction.

We have shown that ex vivo pre-conditioning of bone marrow-derived dendritic cells (DC) with low molecular weight hyaluronan (LMW HA) induces antitumor immunity against colorectal carcinoma (CRC) in mice. In the present study we investigated the effects of LMW HA priming on human-tumor-pulsed monocytes-derived dendritic cells (DC/TL) obtained from healthy donors and patients with CRC. LMW HA treatment resulted in an improved maturation state of DC/TL and an enhanced mixed leucocyte reaction activity in vivo. Importantly, pre-conditioning of DC/TL with LMW HA increased their ability to migrate and reduced their attraction to human tumor derived supernatants. These effects were associated with increased CCR7 expression levels in DC. Indeed, a significant increase in migratory response toward CCL21 was observed in LMW HA primed tumor-pulsed monocyte-derived dendritic cells (DC/TL/LMW HA) when compared to LWM HA untreated cells (DC/TL). Moreover, LMW HA priming modulated other mechanisms implicated in DC migration toward lymph nodes such as the metalloproteinase activity. Furthermore, it also resulted in a significant reduction in DC migratory capacity toward tumor supernatant and IL8 in vitro. Consistently, LMW HA dramatically enhanced in vivo DC recruitment to tumor-regional lymph nodes and reduced DC migration toward tumor tissue. This study shows that LMW HA--a poorly immunogenic molecule--represents a promising candidate to improve human DC maturation protocols in the context of DC-based vaccines development, due to its ability to enhance their immunogenic properties as well as their migratory capacity toward lymph nodes instead of tumors

Diseminación de blaIMP-8 en enterobacterias aisladas en un hospital de Buenos Aires

Entre agosto de 2008 y diciembre de 2011 se detectaron en el Hospital Interzonal General de Agudos «Evita» de Lanús 6 aislamientos de enterobacterias productoras de metalo-ß-lactamasas, distribuidos en tres especies: Enterobacter cloacae (4), Klebsiella oxytoca (1) y Citrobacter freundii (1). Los seis aislamientos presentaron un perfil de multirresistencia y se confirmó la presencia del gen blaIMP-8. Cinco aislamientos además expresaron la ß-lactamasa de espectro extendido PER-2. El gen blaIMP-8 fue hallado como primer casete de un integrón de clase 1. Sin embargo, la secuencia 3´ conservada no pudo detectarse en tres aislamientos. En todos los casos, el gen blaIMP-8 fue transferido por conjugación a Escherichia coli resistente a azida. Mediante PFGE se observó que los cuatro aislamientos de E. cloacae no estuvieron genéticamente relacionados. Estos son los primeros hallazgos de metalo-ß-lactamasas en la institución, que sugieren una posible diseminación horizontal del gen blaIMP-8 intra e interespecies

LMW HA treatment increases migratory response of DC toward CCL21 inducing their CCR7 expression and increases their migratory response toward lymph node supernatant.

<p>a) Migratory ability of DC treated or not with LMW HA toward CCR7 ligand CCL21 (200 ng/ml) was evaluated in a Boyden chamber system. DC were loaded into the upper well of the chamber and CCL21 was added to the lower chamber. Data are expressed as DC/TL and DC/TL/LMW HA migration index. Poly (I:C) was used as a positive control of maturation stimulus. b) CCR7 expression was examined by qPCR. Black bars represent CCR7 fold-change expression in comparison with DC/TL. c) Similar chemotaxis assays were set up placing lymph node supernatant from tumor bearing nude mice in the lower chamber as chemoattractant. Bars represent DC migration index from both HD and CRC patients. Gray bar: DC/TL; black bar: DC/TL/LMW HA; white bar: DC/TL/Poly (I:C). DC/TL vs DC/TL/LMW HA. Mann Whitney t test; * p≤0.05; **p≤0.01.</p

LMW HA induces resistance in human DC to IL-8 chemoattraction.

<p>a) Chemotaxis assays were set up placing IL-8 in the lower chamber and LMW HA treated or untreated DC were placed in the upper chamber. b) A similar migration assay was set up placing TCM with addition of a specific antibody against IL-8 (mAbIL-8) or isotype antibody (20 µg/ml) in the bottom chamber as chemoattractant. Graphs represent data from 4 independent experiments similarly performed expressed as migration index respect to DC/TL migration toward TCM; five fields were counted for each quadruplicate well. Gray bars: DC/LT; black bars: DC/TL/LMW HA; white bars: DC/TL/Poly (I:C). Mann Whitney t test; *p≤0,05; ***p≤0,001. * vs. DC/TL.</p

DC pre-treated with LMW HA induces MMP activity and downregulates HA receptors expression.

<p>a) MMP activity was analyzed by zymography and quantified by densitometry. Relative MMP activity was obtained by normalizing values to untreated samples (DC/TL). Bars represent MMP activity from HD. b) qPCR of HA receptors was performed. The results are expressed as fold change related to DC/TL. Gray bar: DC/TL; black bar: DC/TL/LMW HA. DC/TL vs DC/TL/LMW HA. Mann Whitney t test; *p≤0,05; ***p≤0,001.</p

Pre-incubation of DC with LMW HA increases their migration toward lymph node and reduces their tumor attraction <i>in vivo.</i>

<p>DC/TL were or not LMW HA treated, and after CM DiL labeling were inoculated s.c. in human-tumor bearing nude mice. Twenty four hours later lymph nodes (a) and tumors (b) were surgically removed and a cell suspension was obtained from these tissues. Number of fluorescent DC was counted by flow cytometry. The upper panel (a) shows DC/TL migration toward lymph nodes. The lower panel shows migration toward tumor tissue (b). Bars represent the number of migrated DC per individual HD (1–6) and CRC patients (P 1–4). Gray bar: DC/TL; black bar: DC/TL/LMW HA. * DC/TL vs DC/TL/LMW HA. Paired t test; *p≤0,05.</p

Phenotipic analyses of LMW HA pre-treated DC.

<p>PBMC-derived DC from healthy donors (HD) and colorectal carcinoma (CRC) patients were stained with mAbs anti-CD11c, MHC-II, CD86, CD83 and CD40. CD11c⁺ cells were gated and the co-expression of several markers was analyzed. Data are expressed as geometric mean fluorescence.</p><p>*vs HD DC/LT,</p>δ<p>vs CRC patients DC/TL;</p><p>p≤0,05.</p><p>Phenotipic analyses of LMW HA pre-treated DC.</p