Production of HlyA and ClyA haemolysins among quinolone-resistant Escherichia coli isolated from clinical samples

Most Escherichia coli resistant to quinolones are not haemolytic. The objective of this study was to determine the phylogroup, clonal relationship, mechanism of quinolone resistance and virulence factors in 70 haemolytic E. coli resistant to nalidixic acid. Sixty-six isolates contained the hlyA gene, belonged to phylogroup B2, and 61 of them presented low-level resistance to fluoroquinolones. Four isolates presented high-level resistance to fluoroquinolones, contained the clyA gene and were included in phylogroup D. One single isolate (phylogroup D, with low level resistance to fluoroquinolones) contained both cytotoxins.Supported by Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, Madrid, Spain, co-financed by European Development Regional Fund “A way to achieve Europe” ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015). Alicia Márquez-López was supported by the REIPI and has been supported by a grant from the Instituto de Formación e Investigación Marqués de Valdecilla (IFIMAV), Santander, Spain. We want to thank Eduardo López for his review of English version of the manuscript.S

Optimizing the procedure to manufacture clinical‐grade NK cells for adoptive immunotherapy

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical‐scale. Methods: RPMI‐1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15‐41BBL or K562mbIL21‐41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical‐grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21‐41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21‐41BBL or K562mbIL15‐41BBL. Clinical‐grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP‐grade NK cells for clinical use can be obtained by using different starting cells and aAPCThis work was supported by the National Health Service of Spain, Instituto de Salud Carlos III (ISCIII), FONDOS FEDER grant (FIS) PI18/01301 to Pérez-Martínez A, CRIS Foundation to Beat Cancer to Escudero A, Fernández A; Navarro A, Mirones I, and Fundación Mari Paz Jiménez Casado and La Sonrisa de Álex to Vela

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine ca

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant <i>Staphylococcus aureus</i> (MRSA) Biofilms

<div><p>Ceftaroline (CPT) is a novel cephalosporin with <i>in vitro</i> activity against <i>Staphylococcus aureus</i>. Ceftaroline exhibits a level of binding affinity for PBPs in <i>S</i>. <i>aureus</i> including PBP2a of methicillin-resistant <i>S</i>. <i>aureus</i> (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal <i>icaA</i>, <i>agrA</i>, <i>sarA</i> and <i>sasF</i> genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.</p></div

CLSM of biofilms formed by MRSA strains in presence of ceftaroline.

<p>Confocal Laser Scanning Microscopic images of strains 06/1483, 05/2369, and 05/3291 after growth on uncoated 4-well chamber slides and stained with the LIVE/DEAD viability kit. Live cells are stained green with Syto 9 dye and dead cells are stained red with propidium iodide. MIC and sub-MICs are indicated. Original magnification ×200.</p

FilmMatrix and DNA staining.

<p>(A) coloured proteic matrix of strain 05/3291 exposed to different sub-MICs of ceftaroline. <b>No Ab</b>, without ceftaroline. (B) Biofilm composition of strains 06/1483 and 05/3291. CLSM of Film Tracer (purple) and NucBlue (blue) staining of strain<b>s</b> 06/1483 (left), and 05/3291 (right) without ceftaroline. Original magnification: ×200. Scale bars, 10μm.</p

Effects of ceftaroline on biofilm formation.

<p>Biofilm formation by MRSA in presence of ceftaroline, in 24-well polystyrene plates after 48 h, assessed by crystal violet staining. Each bar indicates the mean values ± SE from four independent experiments. Strains 06/1483, 05/2369, 05/3291 were tested. ATCC 29213 was also included. 0, no antibiotic added (control). X-axis represents ceftaroline concentrations (respect to the MIC). *Indicates significant differences between the concentration tested and control.</p

The bacterial cell morphology of MRSA treated with ceftaroline.

<p>TEM, A-D; SEM, (A´-D´). (A-A´) strain 06/1483 after exposure to 1/2 MIC of ceftaroline. (C-C´) strain 06/1483 without antibiotic. (B-B´) strain 05/3291 after exposure to 1/2 MIC of ceftaroline. (D-D´) strain 05/3291 without antibiotic. Biofilms of strains 06/1483 (E) and 05/3291 (F) exposed to 1/2 MIC of ceftaroline. Original Magnifications: (A) ×80.000; (A´) ×15.000; (B) ×50.000; (B´) ×15.000; (C) ×40.000; (C´) ×20.000; (D) ×60.000; (D´) ×20.000. (E,F) ×15.000. Scale bars, (A-D´) 1μm.</p