Occurrence of blaOXA-23 gene in imipenem-susceptible Acinetobacter baumannii

The aim of the current study was to describe the occurrence of the blaOXA-23 gene and the ISAba1 element in imipenem-susceptible Acinetobacter baumannii strains. By performing the polymerase chain reaction mapping using combinations of ISAba1 forward primers and the blaOXA-23-like gene reverse primers, we demonstrated that the ISAba1 element did not occur upstream of the blaOXA-23 gene in five of 31 isolates, which explained the lack of resistance to imipenem despite the presence of the blaOXA-23 gene. All of the blaOXA-23-positive isolates were susceptible to imipenem and meropenem with minimal inhibitory concentration ≤ 4 μg/mL. Pulsed-field gel electrophoresis analysis revealed four genotypes among the five blaOXA-23-positive isolates. The current report of the blaOXA-23 gene in imipenem-susceptible isolates provided evidence that this gene may be silently spread in a hospital environment and highlighted the threat of undetected reservoirs of carbapenemase genes.     

同时携带blaKPC和blaOXA-10基因的亚胺培南耐药阴沟肠杆菌的研究

目的研究宁波地区临床分离的亚胺培南耐药的阴沟肠杆菌所携带的β-内酰胺酶耐药基因,以确定本地区亚胺培南耐药的阴沟肠杆菌的主要基因型别及其流行情况,指导临床合理用药。方法收集2010年1月1日至2011年4月30日临床分离的阴沟肠杆菌,筛选出亚胺培南耐药菌株用PCR法进行blaOXA-10、blaOXA-2、blaOXA-1、blaKPC、blaIMP、blaVIM、blaNDM-1、blaIMI-1、blaSME、blaSHV-38等多种基因的同时检测。结果共收集到阴沟肠杆菌311株,筛选出对亚胺培南耐药的6株(编号为37号、60号、89号、90号、92号、120号),占1.92%;PCR检测结果显示60号、89号、92号、120号菌株同时产blaKPC型碳青霉烯酶和OXA-10型广谱β-内酰胺酶。结论首次在亚胺培南耐药的阴沟肠杆菌中发现了blaKPC与blaOXA-10同时存在的菌株;亚胺培南耐药的阴沟肠杆菌其耐药机制复杂,且多种耐药机制共存,亟待我们积极深入的探索研究。     

Genome sequences of three Acinetobacter baumannii strains assigned to the multilocus sequence typing genotypes ST2, ST25, and ST78

Acinetobacter baumannii is an emerging opportunistic gram-negative pathogen responsible for hospital-acquired infections. A. baumannii epidemics described in Europe and worldwide were caused by a limited number of genotypic clusters of multidrug-resistant strains. Here, we report the availability of draft genome sequences for three multidrug-resistant A. baumannii strains assigned to multilocus sequence typing genotypes ST2, ST25, and ST78 that were more frequently isolated during outbreaks occurred in Greece, Italy, Lebanon, and Turkey.     

Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I expression in gastric carcinoma cell lines.

Mucin O-glycosylation in cancer is characterized by aberrant expression of immature carbohydrate structures leading to exposure of simple mucin-type carbohydrate antigens and peptide epitopes. Glycosyltransferases controlling the initial steps of mucin O-glycosylation are responsible for the altered glycosylation observed in cancer. We studied the expression in gastric cell lines of six UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T1, T2, T3, T4, T6, T11) that catalyze the initial key step in the regulation of mucin O-glycosylation, the transfer of GalNAc from UDP-GalNAc to serine and threonine residues. We also studied the expression of ST6GalNAc-I, the enzyme responsible for the synthesis of Sialyl-Tn antigen (NeuAcalpha2,6GalNAc) and the ST3Gal-I, the enzyme responsible for the synthesis of Sialyl-T antigen (NeuAcalpha2,3Galbeta1,3GalNAc). This study was done using specific monoclonal antibodies, enzymatic assays, and RT-PCR. Our results showed that GalNAc-T1, -T2, and -T3 have an ubiquitous expression in all gastric cell lines, whereas GalNAc-T4, -T6, and -T11 show a restricted expression pattern. The immunoreactivity with MAb VU-2-G7 suggests that, apart from GalNAc-T4, another GalNAc transferase is involved in the glycosylation of the Thr in the PDTR region of the MUC1 tandem repeat. The expression of ST3Gal-I correlates with the expression of the Sialyl-T antigen in gastric cell lines and in the control cell lines studied. The expression of ST6GalNAc-I is low in gastric cell lines, in accordance with the low/absent expression of the Sialyl-Tn antigen.     

Simple and reliable multiplex PCR assay for detection of blaTEM,bla(SHV) and blaOXA-1 genes in Enterobacteriaceae

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.     

Comparative genomics inferred two distinct populations of piscine pathogenic Streptococcus agalactiae,serotype Ia ST7 and serotype III ST283, in Thailand and Vietnam

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008–2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.     

Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: cloning, expression, characterization, and developmental expression

By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at approximately 6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.     

F(ST) and Q(ST) under neutrality

A commonly used test for natural selection has been to compare population differentiation for neutral molecular loci estimated by F_ST and for the additive genetic component of quantitative traits estimated by Q_ST. Past analytical and empirical studies have led to the conclusion that when averaged over replicate evolutionary histories, Q_ST = F_ST under neutrality. We used analytical and simulation techniques to study the impact of stochastic fluctuation among replicate outcomes of an evolutionary process, or the evolutionary variance, of Q_ST and F_ST for a neutral quantitative trait determined by n unlinked diallelic loci with additive gene action. We studied analytical models of two scenarios. In one, a pair of demes has recently been formed through subdivision of a panmictic population; in the other, a pair of demes has been evolving in allopatry for a long time. A rigorous analysis of these two models showed that in general, it is not necessarily true that mean Q_ST = F_ST (across evolutionary replicates) for a neutral, additive quantitative trait. In addition, we used finite-island model simulations to show there is a strong positive correlation between Q_ST and the difference Q_ST − F_ST because the evolutionary variance of Q_ST is much larger than that of F_ST. If traits with relatively large Q_ST values are preferentially sampled for study, the difference between Q_ST and F_ST will also be large and positive because of this correlation. Many recent studies have used tests of the null hypothesis Q_ST = F_ST to identify diversifying or uniform selection among subpopulations for quantitative traits. Our findings suggest that the distributions of Q_ST and F_ST under the null hypothesis of neutrality will depend on species-specific biology such as the number of subpopulations and the history of subpopulation divergence. In addition, the manner in which researchers select quantitative traits for study may introduce bias into the tests. As a result, researchers must be cautious before concluding that selection is occurring when Q_ST ≠ F_ST.     

Tissue distribution and subcellular localization of a variant form of the human ST2 gene product, ST2V

The human ST2 gene has been known to encode three splice variants; namely, a soluble secreted form of ST2, a transmembrane form of ST2L, and ST2V of undetermined localization. Therefore, analysis of tissue distribution and subcellular localization of ST2V is important to elucidate functional relationships among the three splice variants of the human ST2 gene. RT-PCR procedure revealed that ST2V is predominantly expressed in the stomach, small intestine, and colon. Transfection of ST2V cDNA into COS7 cells in the presence of [(35)S] methionine and cysteine produced radiolabeled 40 kDa protein, which is recognized by specific monoclonal antibody against human ST2. Subcellular fractionation analysis showed that ST2V protein was distributed in the insoluble fraction of the cell lysate. Finally, ST2V protein was detected on the plasma membrane of COS7 cells, which had been transfected with ST2V cDNA, by confocal laser microscopic analysis. These findings taken together, indicate that ST2V protein localizes on the plasma membrane, suggesting its possible role in modification of the ST2L-signaling pathways.     

Adaptive divergence within and between ecotypes of the terrestrial garter snake, Thamnophis elegans, assessed with F(ST)-Q(ST) comparisons

Populations of the terrestrial garter snake (Thamnophis elegans) around Eagle Lake in California exhibit dramatic ecotypic differentiation in life history, colouration and morphology across distances as small as a few kilometres. We assayed the role of selection in ecotypic differentiation in T. elegans using F(ST)-Q(ST) analysis and identified selective agents using direct and indirect observations. We extended the conventional implementation of the F(ST)-Q(ST) approach by using three-level analyses of genetic and phenotypic variance to assess the role of selection in differentiating populations both within and between ecotypes. These results suggest that selection has driven differentiation between as well as within ecotypes, and in the presence of moderate to high gene flow. Our findings are discussed in the context of previous correlational selection analyses which revealed stabilizing and correlational selection for some of the traits examined.     

Optimization of bacteriocin ST311LD production by Enterococcus faecium ST311LD, isolated from spoiled black olives

Bacteriocin ST311LD is approximately 2.3 kDa in size. Low levels of bacteriocin activity were recorded in BHI and M17 broth (800 AU/ml) and in 10% (w/v) soy milk (3,200 AU/ml). No bacteriocin production was recorded in 10% (w/v) molasses, despite good growth. Optimal levels (12,800 AU/ml) were detected in MRS broth which had been supplemented with tryptone (20.0 g/l), saccharose (5.0 or 10.0 g/l) or vitamin C (1 ppm). Increased potassium levels did not result in higher levels of activity, and glycerol (1.0 g/l) inhibited the production of bacteriocin ST311LD.     

Detection of Methicillin-Susceptible Staphylococcus aureus ST398 and ST133 Strains in Gut Microbiota of Healthy Humans in Spain

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n?=?11) and sequences types (n?=?8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4′)-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.     

ST2 and IL-33 in pregnancy and pre-eclampsia

Normal pregnancy is associated with a mild systemic inflammatory response and an immune bias towards type 2 cytokine production, whereas pre-eclampsia is characterized by a more intense inflammatory response, associated with endothelial dysfunction and a type 1 cytokine dominance. Interleukin (IL)-33 is a newly described member of the IL-1 family, which binds its receptor ST2L to induce type 2 cytokines. A soluble variant of ST2 (sST2) acts as a decoy receptor to regulate the activity of IL-33. In this study circulating IL-33 and sST2 were measured in each trimester of normal pregnancy and in women with pre-eclampsia. While IL-33 did not change throughout normal pregnancy, or between non-pregnant, normal pregnant or pre-eclamptic women, sST2 was significantly altered. sST2 was increased in the third trimester of normal pregnancy (p<0.001) and was further increased in pre-eclampsia (p<0.001). This increase was seen prior to the onset of disease (p<0.01). Pre-eclampsia is a disease caused by placental derived factors, and we show that IL-33 and ST2 can be detected in lysates from both normal and pre-eclampsia placentas. ST2, but not IL-33, was identified on the syncytiotrophoblast layer, whereas IL-33 was expressed on perivascular tissue. In an in vitro placental perfusion model, sST2 was secreted by the placenta into the 'maternal' eluate, and placental explants treated with pro-inflammatory cytokines or subjected to hypoxia/reperfusion injury release more sST2, suggesting the origin of at least some of the increased amounts of circulating sST2 in pre-eclamptic women is the placenta. These results suggest that sST2 may play a significant role in pregnancies complicated by pre-eclampsia and increased sST2 could contribute to the type 1 bias seen in this disorder.