Usefulness of High-Quality Core Genome Single-Nucleotide Variant Analysis for Subtyping the Highly Clonal and the Most Prevalent Salmonella enterica Serovar Heidelberg Clone in the Context of Outbreak Investigations.

Salmonella enterica serovar Heidelberg is the second most frequently occurring serovar in Quebec and the third-most prevalent in Canada. Given that conventional pulsed-field gel electrophoresis (PFGE) subtyping for common Salmonella serovars, such as S. Heidelberg, yields identical subtypes for the majority of isolates recovered, public health laboratories are desperate for new subtyping tools to resolve highly clonal S. Heidelberg strains involved in outbreak events. As PFGE was unable to discriminate isolates from three epidemiologically distinct outbreaks in Quebec, this study was conducted to evaluate whole-genome sequencing (WGS) and phylogenetic analysis as an alternative to conventional subtyping tools. Genomes of 46 isolates from 3 Quebec outbreaks (2012, 2013, and 2014) supported by strong epidemiological evidence were sequenced and analyzed using a high-quality core genome single-nucleotide variant (hqSNV) bioinformatics approach (SNV phylogenomics [SNVphyl] pipeline). Outbreaks were indistinguishable by conventional PFGE subtyping, exhibiting the same PFGE pattern (SHEXAI.0001/SHEBNI.0001). Phylogenetic analysis based on hqSNVs extracted from WGS separated the outbreak isolates into three distinct groups, 100% concordant with the epidemiological data. The minimum and maximum number of hqSNVs between isolates from the same outbreak was 0 and 4, respectively, while >59 hqSNVs were measured between 2 previously indistinguishable outbreaks having the same PFGE and phage type, thus corroborating their distinction as separate unrelated outbreaks. This study demonstrates that despite the previously reported high clonality of this serovar, the WGS-based hqSNV approach is a superior typing method, capable of resolving events that were previously indistinguishable using classic subtyping tools.

Molecular epidemiology and antimicrobial susceptibility profiles of methicillin-resistant Staphylococcus aureus blood culture isolates: results of the Quebec Provincial Surveillance Programme.

The objectives of this study were to characterize methicillin-resistant Staphylococcus aureus (MRSA) blood culture isolates and to determine their relative importance in both nosocomial and community-acquired infections. A total of 535 MRSA blood culture isolates were analysed. In vitro susceptibility to 14 agents was determined. The genes nuc, mecA and coding for PVL toxin were identified by PCR. All isolates were characterized by PFGE or spa typing to assess their genomic relationships. Most MRSA isolates were retrieved from nosocomial bloodstream infections (474, 89%) and were of the CMRSA2 genotype. Healthcare-associated (HA)-MRSA bloodstream infections were associated with older age (70-89 years, P = 0·002) and most often secondary to central line infections (P = 0·005). Among MRSA strains associated with community-acquired (CA)-MRSA, 28·8% were isolated in intravenous drug users. CA-MRSA genotypes were more frequently found in young adults (20-39 years, P < 0·0001) with skin/soft tissue as the primary sources of infection (P = 0·006). CMRSA10 genotype was the predominant CA-MRSA strain. All MRSA isolates were susceptible to doxycycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin. Both the presence of the genes coding for PVL toxin (89·8%) and susceptibility to clindamycin (86·5%) were predictive of CA-MRSA genotypes. Whereas in the USA, HA-MRSA have been replaced by USA300 (CMRSA10) clone as the predominant MRSA strain type in positive blood cultures from hospitalized patients, this phenomenon has not been observed in the province of Quebec.

Induction of PG G/H synthase-2 in bovine myometrial cells by interferon-tau requires the activation of the p38 MAPK pathway.

PGs are regulators of a plethora of uterine functions during reproductive processes, including uterine contractility. In bovine uterus, the rate-limiting step in PG synthesis is catalyzed by the PG endoperoxide G/H synthase (PGHS) enzymes. It has previously been established that PGHS-2 isoform expression is affected by the ruminant-specific interferon (IFN)-tau in bovine endometrial cells. Here, we show that PGHS-2 mRNA and protein levels are induced by IFN-tau in primary cell cultures from bovine myometrium. Treatment with recombinant bovine IFN-tau induces the activation of the JAK-STAT and p38 MAPK pathways in bovine myometrial cells. Inhibition of the p38 pathway by the specific inhibitor SB203580 strongly decreases PGHS-2 mRNA and protein expression without affecting the phosphorylation and DNA-binding of transcription factors STAT-1 and STAT-2. The p38 pathway regulates PGHS-2 expression at the posttranscriptional level, because the presence of SB203580 results in the destabilization of IFN-tau-induced PGHS-2 mRNA. Taken together, these data demonstrate the ability of IFN-tau to induce the activation of the JAK-STAT pathway in a manner similar to other types of IFN (i.e. alpha, beta, and gamma) and to regulate PGHS-2 mRNA stability through the activation of the p38 pathway. These findings provide new insights into the physiological function of IFN-tau, in regard to regulation of specific genes associated with myometrial contractility.

Prostaglandin G/H synthase (PGHS)-2 expression in bovine myometrium: influence of steroid hormones and PGHS inhibitors.

Prostaglandins (PGs) are important mediators regulating uterine functions during the reproductive process. The objective of this study was to examine, in myocytes from the circular and longitudinal layers of bovine myometrium, the relative levels of mRNA and proteins corresponding to the gene expression of key enzymes (phospholipase A2; prostaglandin G/H synthase-1 [PGHS-1]; prostaglandin G/H synthase-2 [PGHS-2]; prostaglandin I2 synthase) involved in PG biosynthesis. We examined the influence of estradiol-17beta and progesterone on the expression and activity of these enzymes. Treatment of myocytes with progesterone (P4: 10 nM, 24 h) in the absence or presence of estradiol-17beta (E2: 1 nM, 72 h) suppressed PG biosynthesis by approximately 60% in both myometrial layers. No significant effect was observed after E2 treatment. The combined effect of E2 and P4 on PG accumulation was correlated with the modulation of PGHS-2 protein and mRNA levels in the two myometrial layers without affecting other enzymes of the PG cascade. Selective or nonselective inhibition of PGHS activity with CGP 28238 (PGHS-2-specific; a product from Ciba-Geigy: 6-[2, 4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone) or indomethacin (PGHS-1 and -2) reduced prostacyclin accumulation (measured as 6-keto-PGF1alpha in the culture medium) in a dose-dependent manner in the two myometrial layers. A significant inhibitory effect was obtained at a low concentration of indomethacin (1 nM, p < 0.05) compared to CGP 28238 (10 nM, p < 0. 05). In both myometrial layers, the maximal effect of indomethacin and/or CGP 28238 on PG accumulation was observed at 100 nM and represented 85% and 65% inhibition, respectively. In the presence of phorbol 12-myristate (100 nM), CGP 28238 (10 nM) significantly suppressed PGHS-2 mRNA level by 44.80 +/- 7.67% (p < 0.01) and 27.83 +/- 7.62% (p < 0.05) in the longitudinal and circular layer, respectively. In contrast, indomethacin did not have any significant effect. These data constitute the first quantitative analysis of key enzymes involved in PG biosynthesis in separated myometrial layers. Furthermore, the results provide interesting information on the CGP 28238 drug modulating both enzymatic activity and mRNA expression of PGHS-2.

Down-regulation of interleukin-1beta production and PGE2 accumulation by an indomethacin-phenylalanine derivative in human monocytes.

This study was initiated to investigate the mechanism of action of a new indomethacin derivative, indomethacin-phenylalanine (indo-Phe) in human monocytes. We determined the effect of indo-Phe on the induction by LPS of prostaglandin-E2 (PGE2) and interleukin-1beta (IL-1beta) production in human monocytes. Indomethacin and indo-Phe inhibited the PGE2 synthesis in treated and untreated IL-1beta or LPS-treated monocytes. Furthermore, in IL-1beta and LPS-treated monocytes, prostaglandin G/H synthase-1 (PGHS-1) protein expression was down-regulated with indomethacin or its indo-Phe analog whereas the level of the inducible protein (PGHS-2) was up-regulated. We analyzed the effect of indomethacin and indo-Phe on the expression of IL-1beta protein in LPS-treated monocytes and found that indo-Phe blocked the LPS-induction of IL-1beta synthesis while indomethacin did not. These differential effects of indomethacin and indo-Phe suggest that two independent ways are involved in the stimulation of monocytes by LPS: the PGHS-2 protein induction and the IL-1beta secretion.

Differential expression and regulation of connexin-43 and cell-cell coupling in myocytes from the circular and longitudinal layers of bovine myometrium.

The expression and localization of the gap junction protein connexin-43 (Cx-43) as well as functional coupling were studied in myocytes from the two layers of the bovine myometrium: the circular and the longitudinal layers. Intercellular communication (measured by Lucifer yellow dye transfer through gap junctions) was more intense in the circular than in the longitudinal layer of the bovine myometrium. The circular layer also exhibited a greater degree of punctuate immunofluorescence to Cx-43. Myocytes from the circular layer expressed more Cx-43 messenger RNA (mRNA; 2.38 +/- 0.46, Cx-43 over 18S RNA) than the longitudinal layer (1.46 +/- 0.48, Cx-43 over 18S RNA; P < 0.05). The modulation of Cx-43 expression by sex steroids in the two myometrial layers was tested using a pure steroidal antiestrogen, EM-139. In myocytes from the circular layer, the level of Cx-43 mRNA was decreased after treatment with 0.1 microM EM-139 (1.37 +/- 0.25, Cx-43 over 18S RNA) compared to that in untreated cells (2.38 +/- 0.46, Cx-43 over 18S RNA), representing a 40% inhibition. In parallel, cell-cell coupling and the amount of Cx-43 protein were also reduced after antiestrogen treatment. In contrast, treatment of cells from the longitudinal layer with the antiestrogen did not significantly affect the level of Cx-43 mRNA, protein, or cell-cell coupling. These data demonstrate that Cx-43 protein and mRNA are expressed and regulated differentially in myocytes from the circular and longitudinal layers of bovine myometrium. Furthermore, the circular myometrial layer may represent a preferential target for estrogen regulation of the biochemical and mechanical processes controlling contractility.

Differential effects of a steroidal antiestrogen, EM-139, on prostaglandin and cyclic adenosine 3',5'-monophosphate production in the circular and longitudinal layers of bovine myometrium.

The regulation of prostaglandin (PG) production and cAMP generation was studied in vitro in cultured smooth muscle cells isolated specifically from the circular or longitudinal layers of the bovine myometrium. We found that prostacyclin (PGI2) was the principal PG produced by the myometrium, especially in the longitudinal layer, followed closely by PGE2 and marginally by PGF2 alpha. The PG production (fg/ml, mean +/- SD) in the circular and longitudinal layers was, respectively, PGE2 (424.4 +/- 162.0) > PGI2 (189.5 +/- 19.0) > PGF2 alpha (9.5 +/- 3.0) versus PGI2 (751 +/- 36) > PGE2 (515.7 +/- 94.0) > PGF2 alpha (16.3 +/- 3.0); production was stimulated up to 15-fold 24 h after addition of phorbol 12-myristate (PMA; 100 nM). Hormonal control of PG production was assessed by use of a steroidal antiestrogen, EM-139. PG production was inhibited in a concentration-dependent manner by EM-139 in both circular and longitudinal layers, with maximal inhibition at 1 microM. In parallel studies, chronic treatment with EM-139 resulted in significant increases in isoproterenol-induced cAMP production in both muscle layers, but more especially in the circular layer. This antiestrogenic effect was reversed by addition of 17 beta-estradiol. These results indicate that the two smooth muscle layers of the bovine myometrium have distinct patterns of PG production and that the adenylate cyclase/cAMP response of the circular layer is more sensitive to estrogen modulation. Our findings with a cell culture model of separated myometrial layers provide strategic information for a better understanding of the regulation of uterine contractility during pregnancy.