Carbapenem resistance among Gram-negative isolates collected from patients in ICU and non-ICU hospital wards in Hong Kong: SMART 2017-2020.

OBJECTIVES: The aim of this study was to estimate carbapenem resistance in Pseudomonas aeruginosa and Enterobacterales isolated from infected patients in intensive care unit (ICU) and non-ICU hospital wards in Hong Kong. METHODS: Isolates of Pseudomonas aeruginosa (ICU, n = 35; non-ICU, n = 264) and Enterobacterales (ICU, n = 129; non-ICU, n = 1390) were collected in four Hong Kong hospitals in 2017-2020. Clinical and Laboratory Standards Institute broth microdilution minimum inhibitory concentrations (MICs) were interpreted according to Clinical and Laboratory Standards Institute 2021 M100 breakpoints. ß-lactamase genes were identified in imipenem-, imipenem/relebactam-, and ceftolozane/tazobactam-nonsusceptible isolates. RESULTS: Ceftolozane/tazobactam demonstrated potent in vitro activity against both P. aeruginosa (ICU, 88.6%; non-ICU, 98.5%) and Enterobacterales (96.1%; 97.1%). Percent susceptible values for P. aeruginosa isolates from ICU and non-ICU patients, respectively, were as follows: meropenem (ICU, 74.3%; non-ICU, 84.1%) and imipenem (68.6%; 73.1%). Only 1 of 77 isolates tested for ß-lactamase genes carried a carbapenemase (VIM-2). Percent susceptible values for Enterobacterales isolates from ICU and non-ICU patients were as follows: meropenem (100%; 99.4%), ertapenem (100%; 98.0%), and imipenem (88.4%; 88.6%). A total of 62 Enterobacterales isolates were tested for ß-lactamase genes. Only three isolates carried a carbapenemase gene; two (both Escherichia coli) were metallo-ß-lactamase-positive (both NDM-5), and one (Klebsiella pneumoniae) was OXA-48-like-positive. CONCLUSIONS: Carbapenem-nonsusceptible isolates of P. aeruginosa were common (>15% of isolates). P. aeruginosa percent susceptible values for ceftolozane/tazobactam (97.3% susceptible overall) were ≥14% higher than those for carbapenems in both ICU and non-ICU isolates. Carbapenemases were rare among both P. aeruginosa (one isolate) and Enterobacterales (three isolates). Most Enterobacterales isolates tested from ICU and non-ICU patients in Hong Kong hospitals in 2017-2020 were susceptible to meropenem and ertapenem (≥98%); imipenem was less active (89% susceptible).

Activity of ceftolozane/tazobactam against Gram-negative isolates among different infections in Hong Kong: SMART 2017-2019.

Introduction. Ceftolozane/tazobactam was approved by the Drug Office, Department of Health, Government of the Hong Kong Special Administrative Region in 2017.Hypothesis/Gap Statement. Currently the in vitro activity of ceftolozane/tazobactam against Gram-negative pathogens isolated from patients in Hong Kong is undocumented. It would be prudent to document the activity of ceftolozane/tazobactam against Pseudomonas aeruginosa and Enterobacterales isolated from hospitalized patients in Hong Kong.Aim. To describe the in vitro susceptibility of recent clinical isolates of P. aeruginosa and the two most common Enterobacterales species (Klebsiella pneumoniae, Escherichia coli) cultured from respiratory tract, intra-abdominal, urinary tract and bloodstream infection samples to ceftolozane/tazobactam and other commonly used antimicrobial agents.Methodology. CLSI-defined broth microdilution MICs were determined and interpreted for Gram-negative isolates collected in Hong Kong from 2017 to 2019 by the SMART surveillance programme.Results. For P. aeruginosa, 96.7 % of isolates (n=210) were susceptible to ceftolozane/tazobactam, while susceptibility rates were ≥14 % lower to meropenem (82.9 % susceptible), cefepime (82.4 %), ceftazidime (81.4 %), piperacillin/tazobactam (76.7 %) and levofloxacin (79.5 %). Ceftolozane/tazobactam inhibited 85.7 % of piperacillin/tazobactam-nonsusceptible isolates, 80.6-82.1 % of cefepime-, ceftazidime- or meropenem-nonsusceptible isolates, and 75.9 % of multidrug-resistant (MDR) isolates of P. aeruginosa. For K. pneumoniae, 96.1 % of isolates (n=308) were susceptible to ceftolozane/tazobactam compared with meropenem (99.0 % susceptible), piperacillin/tazobactam (93.8 %), cefepime (85.7 %) and ceftazidime (85.4 %). The majority (88.3 %) of ESBL (extended-spectrum ß-lactamase) non-CRE (carbapenem-resistant Enterobacterales) phenotype isolates of K. pneumoniae were susceptible to ceftolozane/tazobactam, comparable to piperacillin/tazobactam (85.0 %) but lower than meropenem (100 %). For E. coli, 98.5 % of isolates (n=609) were susceptible to ceftolozane/tazobactam compared to meropenem (99.3 % susceptible), piperacillin/tazobactam (96.7 %), ceftazidime (82.3 %) and cefepime (76.5 %). The majority (96.7 %) of ESBL non-CRE phenotype isolates of E. coli were susceptible to ceftolozane/tazobactam, similar to both meropenem (100 %) and piperacillin/tazobactam (94.5 %).Conclusions. Overall, >96 % of clinical isolates of P. aeruginosa, K. pneumoniae and E. coli collected in Hong Kong in 2017-2019 were susceptible to ceftolozane/tazobactam, while the activity of several commonly prescribed ß-lactams was reduced, especially for P. aeruginosa. Continued surveillance of ceftolozane/tazobactam and other agents is warranted.

Stathmin1 plays oncogenic role and is a target of microRNA-223 in gastric cancer.

Stathmin1 (STMN1) is a candidate oncoprotein and prognosis marker in several kinds of cancers. This study was aimed to analyze its expression and biological functions in gastric cancer. The expression of STMN1 was evaluated by qRT-PCR, western blot and immunohistochemistry. The biological function of STMN1 was determined by MTT proliferation assays, monolayer colony formation and cell invasion assays using small interference RNA technique in gastric cancer cell lines. We also explored the regulation of STMN1 expression by microRNA-223. STMN1 was upregulated in gastric cancer cell lines and primary gastric adenocarcinomas. STMN1-positive tumors were more likely to be found in old age group and associated with p53 nuclear expression. In diffuse type gastric adenocarcinomas, STMN1 expression was correlated with age (p = 0.043), T stage (p = 0.004) and lymph node metastasis (p = 0.046). Expression of STMN1 in diffuse type gastric adenocarcinoma was associated with poor disease specific survival by univariate analysis (p = 0.01). STMN1 knockdown in AGS and MKN7 cell lines suppressed proliferation (p<0.001), reduced monolayer colony formation (p<0.001), inhibited cell invasion and migration ability (p<0.001) and induced G1 phase arrest. siSTMN1 could also suppress cell growth in vivo (p<0. 01). We finally confirmed that STMN1 is a putative downstream target of miR-223 in gastric cancer. Our findings supported an oncogenic role of STMN1 in gastric cancer. STMN1 might serve as a prognostic marker and a potential therapeutic target for gastric cancer.

MiR-145 modulates multiple components of the insulin-like growth factor pathway in hepatocellular carcinoma.

Profiling of microRNA expression in human cancers has highlighted downregulation of miR-145 as a common event in epithelial malignancies. Here, we describe recurrent underexpression of miR-145 in hepatocellular carcinoma (HCC) and the identification of a biological pathway by which miR-145 exerts its functional effects in liver tumorigenesis. In a cohort of 80 HCC patients, quantitative reverse transcription polymerase chain reaction corroborated reduced miR-145 expression in 50% of tumors, which also correlated with a shorter disease-free survival of patients. One HCC tumor analyzed with low endogenous miR-145 was propagated as cell line. This in vitro model HKCI-C2 maintained low miR-145 level and upon restoration of miR-145 expression, a consistent inhibitory effect on cell viability and proliferation was readily found. Flow cytometric analysis indicated that miR-145 re-expression could induce G(2)-M cell cycle arrest and apoptosis. Multiple in silico algorithms predicted that miR-145 could target a number of genes along the insulin-like growth factor (IGF) signaling, including insulin receptor substrate (IRS1)-1, IRS2 and insulin-like growth factor 1 receptor. We found protein expression of these putative targets was concordantly downregulated in the presence of miR-145. Luciferase reporter assay further verified direct target association of miR-145 to specific sites of the IRS1 and IRS2 3'-untranslated regions. Subsequent analysis also affirmed miR-145 modulation on the IGF signaling cascade by reducing its downstream mediator, namely the active ß-catenin level. Taken together, our study shows for the first time the pleiotropic effect of miR-145 in targeting multiple components of the oncogenic IGF signaling pathway in HCC.

MiR-222 overexpression confers cell migratory advantages in hepatocellular carcinoma through enhancing AKT signaling.

PURPOSE: This study aims to profile the expressions of 156 microRNAs (miRNA) in hepatocellular carcinoma (HCC) and to characterize the functions of miR-222, the most significantly upregulated candidate identified. EXPERIMENTAL DESIGN: miRNA expression profile in HCC tumors, matching adjacent cirrhotic livers, and cell lines was conducted using quantitative PCR. Common miR-222 upregulations were further validated in a larger cohort of tumors. The functional effects of miR-222 inhibition on HCC cell lines were examined. The downstream modulated pathways and target of miR-222 were investigated by coupling gene expression profiling and pathway analysis, and by in silico prediction, respectively. Luciferase reporter assay was done to confirm target interaction. RESULTS: We identified a 40-miRNA signature that could discriminate tumors from adjacent cirrhotic liver tissue, and further corroborated common miR-222 overexpression in tumors relative to its premalignant counterpart (55.3%; P < 0.0001). Increased miR-222 expression correlated significantly with advanced stage HCC and with the shorter disease-free survival of patients (P < or = 0.01). Inhibition of miR-222 in Hep3B and HKCI-9 significantly retarded cell motility (P < 0.05). Further investigations suggested that AKT signaling was the major pathway influenced by miR-222. A consistent reduction of AKT phosphorylation in Hep3B and HKCI-9 was shown following miR-222 suppression. The protein phosphatase 2A subunit B (PPP2R2A) was predicted as a putative miR-222 target in silico. We found that miR-222 inhibition could augment the tumor protein level and restore luciferase activity in reporter construct containing the PPP2R2A 3' untranslated region (P = 0.0066). CONCLUSIONS: Our study showed that miR-222 overexpression is common in HCC and could confer metastatic potentials in HCC cells, possibly through activating AKT signaling.

MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1.

BACKGROUND & AIMS: Recent studies have emphasized causative links between microRNA (miRNA) deregulations and cancer development. In hepatocellular carcinoma (HCC), information on differentially expressed miRNA remained largely undefined. METHODS: Array-based miRNA profiling was performed on HCC cells that were derived from chronic carriers of hepatitis B virus (HBV) and hepatitis C virus (HCV), and nonviral-associated patients. Specific microRNA (miR)-223 and miR-222 deregulations were verified in an independent series of tumors. The functional effect of miR-223 was examined further. An integrative analysis of messenger RNA (mRNA) array with in silico predictions defined potential downstream targets of miR-223. A luciferase reporter assay was conducted to confirm target association. RESULTS: Distinct up-regulations of miR-222, miR-221, and miR-31, and down-regulations of miR-223, miR-126, and miR-122a were identified. Further investigations suggested the highly deregulated miR-223 and miR-222 could unequivocally distinguish HCC from adjacent nontumoral liver, irrespective of viral associations (P

Isolation and transcript analysis of two-component histidine kinase gene Le.nik1 in Shiitake mushroom, Lentinula edodes.

Le.nik1, a two-component histidine kinase gene of Lentinula edodes, the Shiitake mushroom, was identified. The relationship between this two-component signal transduction system and mushroom development was studied. We used a modified RNA arbitrarily-primed PCR (RAP-PCR) method to isolate Le.nik1 as a differentially expressed gene during L. edodes development. We determined the 6.29kb full-length cDNA sequence of Le.nik1. It had high sequence homology to Neurospora crassa nik1, which encoded a histidine kinase essential for development and osmotic response. In L. edodes, the expression level of Le.nik1 was highest during primordium formation and fruiting body maturation. The transcripts were localized predominantly in the developing hymenophores, or mushroom gills, which may indicate the role of a two-component signal transduction system in cell differentiation during mushroom development. Mannitol stress influenced transcript expression of Le.nik1, suggesting that it may be involved in osmo-sensing and regulation. To our knowledge, this is the first report on the two-component system in mushrooms and the first analysis on the distribution of Le.nik1 transcript in the course of fruiting body formation and in parts of fruiting bodies.

Differential gene expression in hepatopancreas of the shrimp Metapenaeus ensis during ovarian maturation.

Differentially expressed genes were identified in the hepatopancreas of Metapenaeus ensis during ovarian maturation via differential display reverse transcription-polymerase chain reaction (DDRT-PCR). They are G-protein signaling modulator 2 (GPSM2), glutamate carboxypeptide II (GCPII), ligatin, C-type lectin, O-linked N-acetylglucosamine transferase (O-GlcNAc transferase), 1-acylglycerol-3-phosphate O-acyltransferase 4 (AGPAT4), vitellogenin (Vg), and hemocyanin. The hepatopancreas Vg gene identified in this study shows 92% and 49% amino acid sequence homology, respectively, to MeVg1 and MeVg2 previously isolated from this species, suggesting the identification of a new Vg gene in M. ensis. Vg gene expression was highest when the ovary was actively developing. The two metabolic enzymes, O-GlcNAc transferase and AGPAT4, exhibited a similar trend of expression to Vg gene, suggesting their involvement in Vg synthesis. The signal transduction related genes (GPSM2, GCPII, ligatin, and C-type lectin) were highly expressed in the hepatopancreas in the initial phase of maturation. These genes may be important for the signaling in the hepatopancreas for synthesis and mobilization of vitellogenin and nutrients to the developing ovary. The present work provides candidate genes for further investigation on the role of hepatopancreas in shrimp reproduction.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp.

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.