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- Novel flow cytometric screening method for bacterial contamination of red blood cells: a proof-of-principle evaluation. [JOURNAL ARTICLE]
- Transfusion 2013 Dec 4.
Platelet concentrates (PCs) are the major source of transfusion-transmitted bacterial infection but the significantly higher number of transfused red blood cells (RBCs) has resulted in a similar occurrence of possible transfusion-associated bacterial infections as those of recipients transfused with platelets. The aim of this study was adaption of the BactiFlow (BF) flow cytometric rapid PC screening method for the detection of bacterial contamination in RBCs.The BF assay was used to detect and count bacteria based on esterase activity in viable cells in RBCs. The initial sample preparation for RBCs included the lysis of RBCs, followed by enrichment of bacteria by centrifugation and subsequent standard PC sample preparation. Two-hundred RBC units were analyzed by BF and the BacT/Alert automated culture system. Furthermore, RBCs were spiked with eight different bacteria to monitor bacterial growth kinetics.The BF showed an excellent correlation to conventional plate count results after RBC sample processing. The diagnostic sensitivity of the assay was determined to fewer than 500 counts/mL. Analysis of RBC units showed concordant negative results by BF and culture. Growth kinetics of bacteria were successfully monitored using flow cytometry, showing that Yersinia enterocolitica or Serratia liquefaciens had the capability to grow in RBCs to high counts.Our study demonstrates the successful modification and application of a rapid flow cytometric screening method for bacterial contamination in RBCs. The BF assay represents a powerful basic tool to study bacterial contamination, potential screening strategies of RBCs, and the clinical outcome of screened RBCs prepared for transfusion.
- Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels. [JOURNAL ARTICLE]
- Pathology 2013 Dec 1.
We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens.A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance.Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result.Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.
- A molecular scheme for Yersinia enterocolitica patho-serotyping derived from genome-wide analysis. [JOURNAL ARTICLE]
- Int J Med Microbiol 2013 Oct 24.
Yersinia enterocolitica is a food-borne, gastro-intestinal pathogen with world-wide distribution. Only 11 serotypes have been isolated from patients, with O:3, O:9, O:8 and O:5,27 being the serotypes most commonly associated with human yersiniosis. Serotype is an important characteristic of Y. enterocolitica strains, allowing differentiation for epidemiology, diagnosis and phylogeny studies. Conventional serotyping, performed by slide agglutination, is a tedious and laborious procedure whose interpretation tends to be subjective, leading to poor reproducibility. Here we present a PCR-based typing scheme for molecular identification and patho-serotyping of Y. enterocolitica. Genome-wide comparison of Y. enterocolitica sequences allowed analysis of the O-antigen gene clusters of different serotypes, uncovering their formerly unknown genomic locations, and selection of targets for serotype-specific amplification. Two multiplex PCRs and one additional PCR were designed and tested on various reference strains and isolates from different origins. Our genotypic assay proved to be highly specific for identification of Y. enterocolitica species, discrimination between virulent and non-virulent strains, distinguishing the main human-related serotypes, and typing of conventionally untypeable strains. This genotyping scheme could be applied in microbiology laboratories as an alternative or complementary method to the traditional phenotypic assays, providing data for epidemiological studies.
- The Limitations of Pulsed-Field Gel Electrophoresis for Analysis of Yersinia enterocolitica Isolates. [JOURNAL ARTICLE]
- Zoonoses Public Health 2013 Nov 15.
This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI <0.6). For biotypes 2, 3 and 4, 79% or more of isolates of each biotype were of just three different PFGE profiles. Because of this, four known outbreaks of yersiniosis would not have been identified by PFGE analysis. However, a previously unrecognized potential outbreak of yersiniosis caused by biotype 4 isolates was identified on the basis of a rare PFGE genotype with spatial and temporal clustering. We conclude that PFGE has a very limited application to the genotyping of Y. enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence.
- Imaging early pathogenesis of bubonic plague: are neutrophils commandeered for lymphatic transport of bacteria? [Journal Article]
- MBio 2013; 4(6)
ABSTRACTVector-borne infections begin in the dermis when a pathogen is introduced by an arthropod during a blood meal. Several barriers separate an invading pathogen from its replicative niche, including phagocytic cells in the dermis that activate immunity by engulfing would-be pathogens and migrating to the lymph node. In addition, neutrophils circulating in the blood are rapidly recruited when the dermal barriers are penetrated. For flea-borne disease, no insect-encoded immune-suppressive molecules have yet been described that might influence the establishment of infection, leaving the bacteria on their own to defend against the mammalian immune system. Shortly after a flea transmits Yersinia pestis to a mammalian host, the bacteria are transported to the lymph node, where they grow logarithmically and later spread systemically. Even a single cell of Y. pestis can initiate a lethal case of plague. In their article, J. G. Shannon et al. [mBio 4(5):e00170-13, 2013, doi:10.1128/mBio.00170-13] used intravital microscopy to visualize trafficking of Y. pestis in transgenic mice in vivo, which allowed them to examine interactions between bacteria and specific immune cells. Bacteria appeared to preferentially interact with neutrophils but had no detectable interactions with dendritic cells. These findings suggest that Y. pestis infection of neutrophils not only prevents their activation but may even result in their return to circulation and migration to distal sites.
- The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo. [Journal Article]
- Microbiologyopen 2013 Dec; 2(6):962-75.
Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt - an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice.
- Economics. Women, fertility, and the rise of modern capitalism. [Historical Article, Journal Article]
- Science 2013 Oct 25; 342(6157):427-8.
- Rare zoonoses in the USA. [News]
- Lancet Infect Dis 2013 Sep; 13(9):740-1.
- Yersinia enterocolitica infections associated with improperly pasteurized milk products: southwest Pennsylvania, March-August, 2011. [JOURNAL ARTICLE]
- Epidemiol Infect 2013 Oct 16.:1-11.
SUMMARY In July 2011, a cluster of Yersinia enterocolitica infections was detected in southwestern Pennsylvania, USA. We investigated the outbreak's source and scope in order to prevent further transmission. Twenty-two persons were diagnosed with yersiniosis; 16 of whom reported consuming pasteurized dairy products from dairy A. Pasteurized milk and food samples were collected from this dairy. Y. enterocolitica was isolated from two products. Isolates from both food samples and available clinical isolates from nine dairy A consumers were indistinguishable by pulsed-field gel electrophoresis. Environmental and microbiological investigations were performed at dairy A and pasteurization deficiencies were noted. Because consumption of pasteurized milk is common and outbreaks have the potential to become large, public health interventions such as consumer advisories or closure of the dairy must be implemented quickly to prevent additional cases if epidemiological or laboratory evidence implicates pasteurized milk as the outbreak source.
- An ironic case of liver infections: Yersinia enterocolitis in the setting of thalassemia. [Journal Article]
- World J Gastroenterol 2013 Oct 7; 19(37):6296-8.
A 49 years old Vietnamese male with a history of thalassemia, presented with gastrointestinal symptoms and signs of hemolysis. He was diagnosed with yersinia enterocolitis. Yersinia is a gram-negative rod that most frequently occurs in children especially during the winter months. In the current case, the bone marrow biopsy showed hemophagocytosis along with positive cultures for Yersinia. The microorganism likely triggered hemophagocytosis. This syndrome, also known as, hemophagocytic lymphohistiocytosis, is defined by fever for more than 7 d, cytopenia of two or more cell lines, hemophagocytosis, hepatitis, serum ferritin greater than 500, jaundice, lymphadenopathy, and hepatosplenomegaly. This disorder can be either familial or secondary to a strong immunologic activation. Both have an overwhelming activation of T-cells and macrophages.