NETs under scanning electron microscopy.

<p>NETs induced by PMA (A and B), cKP (3 isolates, C and D), and HvKP-K1 (3 isolates, E and F). More cKP (C) than HvKP-K1 (E) were trapped in NETs by magnification of 5K. The pores (indicated by white arrows) on the surface of cKP, but not on the surface of HvKP-K1 were observed by magnification of 20K (D and F). Bacteria were indicated by black arrows.</p

Phagocytosis of neutrophils against HvKP-K1, HvKP-K2 and cKP.

<p>The rate of phagocytosis against cKP (15 isolates) was higher than that against HvKP-K1 (16 isolates) or HvKP-K2 (14 isolates) at 10, 30, 60 min. The mean ± standard deviation (S.D.) of each group at each time point was calculated respectively. Statistics was performed using one-way analysis of variance for each time point. Differences between groups were assessed by <i>t</i> test. At 10, 30, 60 min, HvKP-K1 vs. cKP or HvKP-K2 vs. cKP: p < 0.05.</p

Survival of cKP, HvKP-K1 and HvKP-K2 within human neutrophils.

<p>The survival index was calculated with the equation described in the method. The survival index of cKP (15 isolates) was lower than that of HvKP-K1 (16 isolates) or HvKP-K2 (14 isolates). Each strain was repeated twice and averaged. Then the mean ± standard deviation (S.D.) of each group was calculated. Statistics was performed using one-way analysis of variance. Differences between groups were assessed by <i>t</i> test. HvKP-K1 vs. cKP or HvKP-K2 vs. cKP: p < 0.001.</p

Immunofluorescence staining under confocal microscopy.

<p>Neutrophils challenged by cKP (9 isolates, A-D) and HvKP-K1 (9 isolates, E-H) were stained for DNA (DAPI, blue, A and E), myeloperoxidase (MPO, red, B and F) and citrullinated histone H3 (cit-H3, green, C and G). The merged images of cKP (D) and HvKP-K1 (H) illustrated the characteristic neutrophil extracellular traps. Original magnification 40×.</p

A Locked Nucleic Acid (LNA)-Based Real-Time PCR Assay for the Rapid Detection of Multiple Bacterial Antibiotic Resistance Genes Directly from Positive Blood Culture

<div><p>Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.</p></div

Analysis of positive blood culture samples by real-time PCR.

<p>Note: the positive blood culture samples containing <i>P</i>. <i>aeruginosa</i> are not included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120464#pone.0120464.t004" target="_blank">Table 4</a>.</p><p>^a: ß-lactamase: <i>bla</i>_CTX-M-1/<i>bla</i>_CTX-M-9 ESBLs or <i>bla</i>_CMY-2/<i>bla</i>_DHA-1 pAmpC gene.</p><p>^b: Eco, <i>Escherichia coli</i>; Kpn, <i>Klebsiella pneumoniae</i>; Pae: <i>Pseudomonas aeruginosa</i>; Sau, <i>Staphylococcus aureus</i>; Sep, <i>S</i>. <i>epidermidis</i>; Sha, <i>S</i>. <i>haemolyticus</i>; Sho, <i>S</i>. <i>hominis</i>, Efm, <i>Enterococcus faecium</i>; Efs, <i>E</i>. <i>faecalis</i>; and Staph, <i>Staphylococcus</i>.</p><p>^c: <i>van</i>A and <i>van</i>B were not detected in the collected cultures.</p><p>Analysis of positive blood culture samples by real-time PCR.</p

Sequences of primers and LNA probes.

<p>^a: The sequence of the LNA probe was completely shown and all the nucleotides in the LNA probe were LNA nucleotides.</p><p>Sequences of primers and LNA probes.</p

Genes associated with Antimicrobial resistance in BJAB07104, BJAB0868 and BJAB0715.

a<p>R: Ser-Leu mutation at 83, S: no mutation at 83; ^b: R: Ser-Leu mutation at 84, S: no mutation at 84.</p

Complete Genome Analysis of Three <i>Acinetobacter baumannii</i> Clinical Isolates in China for Insight into the Diversification of Drug Resistance Elements

<div><p>Background</p><p>The emergence and rapid spreading of multidrug-resistant <i>Acinetobacter baumannii</i> strains has become a major health threat worldwide. To better understand the genetic recombination related with the acquisition of drug-resistant elements during bacterial infection, we performed complete genome analysis on three newly isolated multidrug-resistant <i>A. baumannii</i> strains from Beijing using next-generation sequencing technology.</p><p>Methodologies/Principal Findings</p><p>Whole genome comparison revealed that all 3 strains share some common drug resistant elements including carbapenem-resistant <i>bla</i>_OXA-23 and tetracycline (<i>tet</i>) resistance islands, but the genome structures are diversified among strains. Various genomic islands intersperse on the genome with transposons and insertions, reflecting the recombination flexibility during the acquisition of the resistant elements. The blood-isolated BJAB07104 and ascites-isolated BJAB0868 exhibit high similarity on their genome structure with most of the global clone II strains, suggesting these two strains belong to the dominant outbreak strains prevalent worldwide. A large resistance island (RI) of about 121-kb, carrying a cluster of resistance-related genes, was inserted into the <i>ATPase</i> gene on BJAB07104 and BJAB0868 genomes. A 78-kb insertion element carrying <i>tra</i>-locus and <i>bla</i>_OXA-23 island, can be either inserted into one of the <i>tniB</i> gene in the 121-kb RI on the chromosome, or transformed to conjugative plasmid in the two BJAB strains. The third strains of this study, BJAB0715, which was isolated from spinal fluid, exhibit much more divergence compared with above two strains. It harbors multiple drug-resistance elements including a truncated AbaR-22-like RI on its genome. One of the unique features of this strain is that it carries both <i>bla</i>_OXA-23 and <i>bla</i>_OXA-58 genes on its genome. Besides, an <i>Acinetobacter lwoffii</i> adeABC efflux element was found inserted into the ATPase position in BJAB0715.</p><p>Conclusions</p><p>Our comparative analysis on currently completed <i>Acinetobacter baumannii</i> genomes revealed extensive and dynamic genome organizations, which may facilitate the bacteria to acquire drug-resistance elements into their genomes.</p></div

Structure of resistance islands AbaR25-27 containing drug resistant gene<i>tetA</i>.

<p>(a) AbaR25 in BJAB07104 and AbaR26 in BJAB0868 are both inserted into the <i>ATPase</i> gene. Transposons Tn6206-6209 are showed in dashed rectangles. While Tn6208 (in BJAB07104) is flanked by two ISAba1 elements, Tn6209 (in BJAB0868) only has the left flanking ISAba1 element. When being present in chromosome, Tn6206 and <i>tra</i> system are inserted into the gene <i>tniB</i>, while this region can also form free plasmids. (b) AbaR27 in BJAB0715 is inserted into gene <i>EJP43116</i>, which produces a protein 100% identical to a hypothetical protein found in <i>A. baumannii</i> OIFC032 strain.</p