BackgroundThe emergence of plasmid-mediated carbapenemases, such as NDM-1 in Enterobacteriaceae is a major public health issue. Since they mediate resistance to virtually all β-lactam antibiotics and there is often co-resistance to other antibiotic classes, the therapeutic options for infections caused by these organisms are very limited.MethodologyWe characterized the first NDM-1 producing E. coli isolate recovered in Hong Kong. The plasmid encoding the metallo-β-lactamase gene was sequenced.Principal FindingsThe plasmid, pNDM-HK readily transferred to E. coli J53 at high frequencies. It belongs to the broad host range IncL/M incompatibility group and is 88803 bp in size. Sequence alignment showed that pNDM-HK has a 55 kb backbone which shared 97% homology with pEL60 originating from the plant pathogen, Erwina amylovora in Lebanon and a 28.9 kb variable region. The plasmid backbone includes the mucAB genes mediating ultraviolet light resistance. The 28.9 kb region has a composite transposon-like structure which includes intact or truncated genes associated with resistance to β-lactams (bla TEM-1, bla NDM-1, Δbla DHA-1), aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD, ΔtnpATn1 and insL. Certain blocks within the 28.9 kb variable region had homology with the corresponding sequences in the widely disseminated plasmids, pCTX-M3, pMUR050 and pKP048 originating from bacteria in Poland in 1996, in Spain in 2002 and in China in 2006, respectively.SignificanceThe genetic support of NDM-1 gene suggests that it has evolved through complex pathways. The association with broad host range plasmid and multiple mobile genetic elements explain its observed horizontal mobility in multiple bacterial taxa.

Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.

Increased serum haptoglobin concentration and changes in its glycosylation have been reported in certain cancer types. Information for hepatocellular carcinoma (HCC) has not yet been available. In this study, we aimed to carry out a systematic analysis of serum concentrations of haptoglobin (Hp) and its glycoforms in the patients with HCC and noncancer patients only with chronic liver diseases (CLD) and to examine their clinical values. This study was divided into two major parts, (1) measurement of serum Hp concentration, and investigation of its value in the diagnosis of HCC, and (2) quantitative analysis of Hp glycoforms with alpha-2,6-sialylation and/or alpha-1,6-fucosylation by using lectin affinity purification and 2D gel electrophoresis and investigation of their relationships with tumor stage. The concentrations of serum Hp in HCC patients were significantly higher than those in noncancer patients with CLD. With the use of serum concentrations of Hp and alpha-fetoprotein, a logistic regression (LR) model was developed from the training data set and used to classify the validation cases. At a specificity of 95%, the sensitivity for HCC detection was 79%. Comparing serum concentrations of alpha-2,6-sialylated Hp (S-Hp) and alpha-1,6-fucosylated Hp (F-Hp) between HCC and CLD patients suggests that purification of S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. 2D gel analysis of S-Hp and F-Hp identified a total of 18 glycoforms. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species was found in the HCC patients. Serum concentrations of these glycoproteins were significantly higher in the patients with advanced tumors, suggesting their tumor-specific nature. We have shown that serum Hp is a potential biomarker in the diagnosis of HCC. The combined use of Hp and AFP could greatly improve the diagnostic accuracy. A unique pattern of Hp glycoforms with altered sialylation and fucosylation is specific to HCC and associated tumor progression.

Fragmentation reactions of protonated α-amino acids (AAs) were studied previously using tandem mass spectrometry (MS/MS) of unit mass resolution. Isobaric fragmentation products and minor fragmentation products could have been overlooked or misannotated. In the present study, we examined the fragmentation patterns of 19 AAs using high-resolution electrospray ionization MS/MS (HR-ESI-MS/MS) with collision-induced dissociation (CID). Isobaric fragmentation products from protonated Met and Trp were resolved and identified for the first time. Previously unreported fragmentation products from protonated Met, Cys, Gln, Arg, and Lys were observed. Additionally, the chemical identity of a fragmentation product from protonated Trp that was incorrectly annotated in previous investigations was corrected. All previously unreported fragmentation products and reactions were verified by pseudo MS 3 experiments and/or MS/MS analyses of deuterated AAs. Clearer pictures of the fragmentation reactions for Met, Cys, Trp, Gln, Arg and Lys were obtained in the present study.

Background:Most noninvasive predictive models of liver fibrosis are complicated and have suboptimal sensitivity. This study was designed to identify serum proteomic signatures associated with liver fibrosis and to develop a proteome-based fingerprinting model for prediction of liver fibrosis. Methods: Serum proteins from 46 patients with chronic hepatitis B (CHB) were profiled quantitatively on surface-enhanced laser desorption/ionization (SELDI) ProteinChip arrays. The identified liver fibrosis-associated proteomic fingerprint was used to construct an artificial neural network (ANN) model that produced a fibrosis index with a range of 0 -6. The clinical value of this index was evaluated by leave-one-out cross-validation. Results: Thirty SELDI proteomic features were significantly associated with the degree of fibrosis. Cross-validation showed that the ANN fibrosis indices derived from the proteomic fingerprint strongly correlated with Ishak scores (r ‫؍‬ 0.831) and were significantly different among stages of fibrosis. ROC curve areas in predicting significant fibrosis (Ishak score >3) and cirrhosis (Ishak score >5) were 0.906 and 0.921, respectively. At 89% specificity, the sensitivity of the ANN fibrosis index in predicting fibrosis was 89%. The sensitivity for prediction increased with degree of fibrosis, achieving 100% for patients with Ishak scores >4. The accuracy for prediction of cirrhosis was also 89%. Inclusion of International Normalized Ratio, total protein, bilirubin, alanine transaminase, and

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Streptococcus pneumoniae serogroup 19 clonal complex 320/271 (CC320/271) includes important multidrug-resistant (MDR) clones, including sequence type 271(ST271), responsible for both invasive pneumococcal disease (IPD) and noninvasive pneumococcal disease in most Asian countries (1, 2) and in parts of China (3). In the United States and other western countries, serotype 19A-ST320, a multidrug-resistant (MDR) clone, has been highly successful after the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) (4). ST320 is a double-locus variant (DLV) of the originally described Taiwan 19F -14 (Pneumococcal Molecular Epidemiology Network; PMEN14) clone, ST236, from isolates from a Taiwanese hospital in 1997 (5) and has undergone a capsular switch to nonvaccine serotype 19A, likely through recombination that altered the wzy polymerase gene. In mainland China, 43.8% of cases of IPD were reportedly due to serogroup 19 during 2005 to 2011, with 84.7% of isolates belonging to CC320/ 271, including ST271, ST320, and ST236 (3). In Hong Kong, we reported the introduction of the Taiwan 19F -14 (ST236) clone back in 1999 (6), prior to the introduction of PCV7. These strains were resistant to ␤-lactams, tetracyclines, and macrolides but susceptible to chloramphenicol and replaced the predominant MDR Spain 23F -1 and Spain 6B -2 clones in the late 1990s (7). The Hong Kong Special Administrative Region (HKSAR) introduced PCV7 to the childhood immunization program in October 2009 and subsequently introduced the 10-valent vaccine in October 2010 and PCV13 in 2011 in keeping with the changes in the vaccine coverage of prevalent serotypes. Of the serotypes included in PCV7, serotype 19F is the least likely to evoke a protective immune response according to both vaccine efficacy trials and in vivo studies (8, 9). Therefore, despite the initial decrease in carriage and invasive disease due to serotype 19F, reemergence of the disease might be anticipated due to a weaning of antibody responses over time. This is reflected by recent studies in Hong Kong comparing pre-and postvaccination serotype carriage, where a significant difference was not found for serotype 19F (10). In addition, during this period, an increasing level of resistance of S. pneumoniae to the third-generation cephalosporin cefotaxime was observed. Given the ability of pneumococci to adapt and transform, we sought to examine the levels of antimicrobial and ␤-lactam resistance of these isolates and study the polymorphisms of amino acid sequences of penicillin binding protein 1a (PBP1a), PBP2b, and PBP2x that might be related to different levels of

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