Amino acid and nucleotide sequence, adjacent genes, and heterologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae DCH5, isolated from Mallard ducks (Anas platyrhynchos)

The primary structure of a bacteriocin produced by Enterococcus hirae DCH5 was determined by combined amino acid and DNA sequencing. Nucleotide analysis of a 2838-bp DNA fragment of E. hirae DCH5 revealed five putative ORFs. The first orf (hirJM79) encodes a 74-amino-acid peptide containing an N-terminal signal peptide of 30 amino acids, followed by the amino acid sequence of the mature bacteriocin, hiracin JM79 (HirJM79), of 44 amino acids. The second orf (hiriJM79) encodes the putative immunity protein of HirJM79. Contiguous ORFs encode a putative mobilization protein (orfC), a relaxase/mobilization nuclease domain (orfD), and a hypothetical protein (orfE). The production and functional expression of HirJM79 by heterologous hosts suggest that hirJM79 is the minimum requirement for production of biologically active HirJM79, that HirJM79 is most likely externalized by the general secretory pathway or sec-dependent pathway, and that HiriJM79 is the immunity protein for HirJM79.     

Isolation of New Delhi metallo‐β‐lactamase 1‐producing Vibrio cholerae non‐O1, non‐O139 strain carrying ctxA,st and hly genes in southern Vietnam

Vibrio cholerae non‐O1, non‐O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non‐pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG‐specific heat‐stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010–2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo‐β‐lactamase (NDM‐1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM‐1 and the bla_NDM‐1 gene was detected in those strains by PCR. Of note, one of the three NDM‐1‐producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM‐1‐producing VC_NAG strains in Vietnam.     

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.     

Kinetics of the early subcellular distribution of cadmium in rat hepatocytes

The kinetics of the early subcellular distribution of cadmium (Cd) was characterized in primary cultures of rat hepatocytes exposed to 10, 50 and 100 M Cd in a serum-free WME medium for 10, 30 or 60 min. Our results demonstrate a time- and concentration-dependent increase in Cd content with the highest metal concentration measured in the cytosol, whereas the lowest was observed in the mitochondria. With the exception of early localization in the plasma membrane, Cd concentrations in fractions were characterized by the following decreasing order of magnitude: cytosol > low density molecules nuclei > lysosomes mitochondria. We also found evidence for: (i) a two-step process for Cd distribution in the nuclei and mitochondria; and (ii) a time-dependent slow process of transfer from the plasma membrane to the cytosol. Saturation in Cd uptake was observed at 50 M in most cell fractions at 10 and 30 min, except for the plasma membrane. The lack of apparent saturation for Cd accumulation at 60 min was not related to an increase in metallothionein synthesis. Altogether, our data provide insights into the dynamics of transfer between intracellular compartments, and allow a better identification of the organelles that are the most subjected to Cd toxicity for early exposure conditions.Deceased March 25, 2004.     

Characterization of a new syndrome that associates craniosynostosis, delayed fontanel closure, parietal foramina, imperforate anus, and skin eruption: CDAGS

We describe the clinical characterization, molecular analyses, and genetic mapping of a distinct genetic condition characterized by craniosynostosis, delayed closure of the fontanel, cranial defects, clavicular hypoplasia, anal and genitourinary malformations, and skin eruption. We have identified seven patients with this phenotype in four families from different geographic regions and ethnic backgrounds. This is an autosomal recessive condition that brings together apparently opposing pathophysiologic and developmental processes, including accelerated suture closure and delayed ossification. Selected candidate genes--including RUNX2, CBFB, MSX2, ALX4, TWIST1, and RECQL4--were screened for mutations, by direct sequencing of their coding regions, and for microdeletions, by fluorescent in situ hybridization. No mutations or microdeletions were detected in any of the genes analyzed. A genomewide screen yielded the maximum estimated LOD score of +2.38 for markers D22S283 and D22S274 on chromosome 22q12-q13. We hypothesize that the gene defect in this condition causes novel context-dependent dysregulation of multiple signaling pathways, including RUNX2, during osteoblast differentiation and craniofacial morphogenesis.     

Long-term effects of the PPAR gamma activator pioglitazone on cardiac inflammation in stroke-prone spontaneously hypertensive rats

We investigated the long-term effects of the thiazolidinedione PPARgamma activator pioglitazone on cardiac inflammation in stroke-prone spontaneously hypertensive rats (SHRSP), a model of malignant of hypertension. Six-week-old SHRSP were treated with pioglitazone (10 mg/kg per day p.o.) for 20 weeks. The rise in systolic blood pressure (SBP) in SHRSP was only transiently and slightly attenuated by pioglitazone (P < 0.05). On one hand, cardiac hypertrophy was little affected by the pioglitazone treatment, and there was only a reduction of subepicardial interstitial fibrosis. On the other hand, left ventricular NFkappaB and AP-1 binding activities, the expression of TNFalpha, and the adhesion of molecule PECAM were significantly decreased by pioglitazone treatment. Expression of the pro-apoptotic proteins p53 and bax was significantly increased by pioglitazone. Thus, pioglitazone-attenuated cardiac inflammation in SHRSP had little effect on BP or cardiac hypertrophy. PPARgamma activation may play a preventive cardiovascular role by offsetting the cardiac inflammatory response as demonstrated in this genetic model of malignant hypertension.     

Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection

Background  

Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specifiCity aspects.     

Construction and Application of a luxABCDE Reporter System for Real-Time Monitoring of Enterococcus faecalis Gene Expression and Growth

The present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging of Enterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains of E. faecalis were constructed. pSL101 harbors the luxABCDE operon from pPL2lux and the pREG696 broad-host-range replicon and axe-txe toxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R(2) > 0.98) with the viable-cell count. We employed lux-tagged E. faecalis strains to monitor growth in real time in milk and urine in vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in the Galleria mellonella model system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI of E. faecalis and an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.     

Genome Sequence of the Bacteriocin-Producing Strain Lactococcus garvieae DCC43

This work describes the draft genome sequence of Lactococcus garvieae DCC43. The 2.2-Mb draft genome contains 2,227 predicted protein-coding genes, among which is a region encoding the bacteriocin garvicin ML. No antibiotic resistance genes or capsule-related virulence genes were identified. Two plasmid replication regions indicate that this strain likely contains plasmids. Comparative genomics suggests that this strain displays a high degree of sequence variation from the previously sequenced L. garvieae strains.