Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when grown on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E. coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation.

Genotypic and phenotypic properties of coagulase-negative staphylococci causing dialysis catheter-related sepsis

Sixty coagulase-negative staphylococcus (CNS) isolates were recovered from the blood cultures or peritoneal dialysate effluent of 43 patients on renal dialysis. The patients had either renal dialysis catheter-related sepsis (CRS) or continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis. Isolates were characterized by biotyping, and genotyped by pulsed-field gel electrophoresis (PFGE). Phenotypic properties of the strains were also investigated. Several genotypes were identified with no one specific strain of CNS being associated with CRS. However, closely related strains were isolated from several patients within the units studied, suggesting horizontal transfer of micro-organisms. Genotypic macro-restriction profiles did not concur with phenotypic profiles or biotypes, confirming that genotyping is required for epidemiological studies. All staphylococcal strains were investigated for the production of phenotypic characteristics. Significant differences were predominantly seen in the production of lipase, esterase and elastase in strains isolated from the renal patients with CRS and CAPD-associated peritonitis, compared with a non-septic control group. These phenotypic characteristics may therefore have a role in the maintenance of CRS in renal patients. © 2003 The Hospital Infection Society. Published by Elsevier Science Ltd. All rights reserved.

A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

Serodiagnosis of staphylococcal sepsis

The coagulase-negative staphylococci are the most frequent cause of sepsis associated with indwelling intravascular catheters. Current microbiological investigations to support the diagnosis of catheter-related sepsis (CRS) include the culture of blood and catheter tips, however positive results may reflect specimen contamination, or colonisation of the catheter rather than true sepsis. Previous serological approaches to assist in the diagnosis of CRS based on cellular staphylococcal antigens have been of limited value. In this current study, the serodiagnostic potential of an exocellular antigen produced by 7 strains of coagulase-negative staphylococci cultured in brain heart infusion broth was investigated. Antigenic material isolated by gel permeation from liquid culture was characterised by immunological techniques and chemical analysis. Characterisation of the exocellular antigen revealed a novel glycerophosphoglycolipid, termed lipid S. which shared antigenic determinants with lipoteichoic acid, but differed by comprising a glycerophosphate chain length of only 6 units. In addition, lipid S was immunologically distinct from diphosphatidyl glycerol, a constituent cell membrane phospho lipid. An indirect enzyme linked immunosorbent assay (ELISA) based on lipid S was subsequently developed and used to determine serum antibody levels (IgM and IgG) in 67 patients with CRS due to staphylococci, and 67 patients with a central venous catheter (CVC) in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the lipid S IgG ELISA was 75% and 90% respectively whilst the IgM assay had sensitivity and specificity of 52% and 85%. The addition of GullSORereagent to the EL1SA procedure to remove competing serum IgG and rheumatoid factor did not significantly improve the performance of the IgM assay. The serological response in serial serum samples of 13 patients with CRS due to staphylococci was investigated. Elevated levels of antibody were detected at an early stage of infection, prior to the isolation of microorganisms by standard culture methods, and before the clinical presentation of sepsis in 3 patients. The lipid S ELISA was later optimised and a rapid 4-hour assay developed for the serodiagnosis of CRS. Serum IgG levels were determined in 40 patients with CRS due to staphylococci and 40 patients with a CVC in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the rapid IgG assay was 70% and 100% respectively. Elevated serum antibody levels in patients with endocarditis, prosthetic joint infection and pyogenic spondylodiscitis due to Gram-positive cocci were also detected with the lipid S ELISA suggesting that the assay may facilitate the diagnosis of these infections. Unexpected increased levels of anti-lipid S IgG in 31% of control patients with sciatica suggested a possible microbial aetiology of this condition. Further investigation of some of these patients by culture of microdiscectomy tissue removed at operation, revealed the presence of low-virulent microorganisms in 37% of patients of which Propionibacterium aeries accounted for 85% of the positive culture isolates. The results suggested a previously unrecognised association between P. acnes and sciatica, which may have implications for the future management of the condition.

Influence of iron deprivation and sub-inhibitory concentrations of antifungal antibiotics on surface antigens of candida albicans yeast cells

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

Studies of skin antisepsis and enhanced penetration of chlorhexidine

Current evidence-based guidelines recommend that 2% (w/v) chlorhexidine digluconate (CHG), preferentially in 70% (v/v) isopropyl alcohol (IIPA), is used for skin antisepsis prior to incision of the skin. In this current study, the antimicrobial efficacy of CHG, six essential oils [tea tree oil (TTO), thymol, eucalyptus oil (EO), juniper oil, lavender oil and citronella] and novel benzylidenecarboxamidrazone and thiosemicarbazone compounds were determined against a panel of microorganisms commonly associated with skin infection (Staphylococcus epidermidis, S. aureus, meticillin-resistant S. aureus, Propionibacterium acnes, Acinetobacter spp., Pseudomonas aeruginosa and Candida albicans) The results demonstrated synergistic activity of CHG in combination with EO against biofilm cultures of S. epidermidis, with significantly reduced concentrations of CHG and EO required to inhibit biofilm growth compared to CHG or EO alone. Skin permeation of CHG was subsequently investigated using an in vitro human skin model (Franz cell) and the penetration profile was determined by serial sectioning of the full thickness human skin. Two percent (w/v) CHG in aqueous solution and in 70% (v/v) IPA demonstrated poor skin permeation; however, the skin permeation was significantly enhanced in combination with 5% - 50% (v/v) EO. Detectable levels of CHG did not permeate through full thickness skin in 24 h. Skin permeation of 2% (w/v) CHG in 70% (v/v) IPA in the presence of 10% (v/v) EO was subsequently studied. The results demonstrated a significantly enhanced skin penetration of CHG after a 2 min application, with CHG detected at significant levels to a depth of 600 m with CHG in combination with EO and IPA compared to 100 m with IPA alone. Combination antisepsis comprising CHG and EO may be beneficial for skin antisepsis prior to invasive procedures to reduce the number of microorganisms on and within the skin due to enhanced skin penetration of CHG and improved efficacy against S. epidermidis in a biofilm mode of growth.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection

Objectives: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). Methods: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. Results: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. Conclusion: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4 h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections. © 2005 Published by Elsevier Ltd on behalf of the British Infection Society.

Logistic Regression in Modelling Data for CVC - Related Infection

2000 Mathematics Subject Classification: 62J12, 62P10.

Addition of PLA2 to CRP enhances sepsis diagnosis

Letter to the Editor refers to: Fredrikke Christie Knudtzen, Stig Lønberg Nielsen, Kim Oren Gradel, Annmarie Touborg Lassen, Hans Jørn Kolmos, Thøger Gorm Jensen, Pernille Just Vinholt, Court Pedersen, Characteristics of patients with community-acquired bacteremia who have low levels of C-reactive protein (≤20 mg/L), Journal of Infection, Volume 68, Issue 2, February 2014, Pages 149-155

Capacidade de adesão e formação de biofilme de Candida ssp. isoladas da cavidade oral de pacientes transplantados renais na presença do extrato de Eugenia uniflora

Candidiasis is a major oral manifestation in kidney transplant patients. Candida spp. possess essential virulence factors which contribute for the infectious process, including the ability to adhere to epithelial cells and biofilm formation. The extract obtained from the leaves of Eugenia uniflora [acetone: water (7:3, v/v)] has demonstrated antifungal activity against Candida spp. This study evaluated the influence of the extract of E. uniflora in adhesion to human buccal epithelial cells (HBEC) and biofilm formation of 42 strains of Candida spp. isolated from the oral cavity of kidney transplant patients. Candida spp. strains belonging to a culture collection were reactivated and phenotypically re-identified by classical and molecular methods (genotyping ABC and RAPD), when necessary, to complete the identification to the species level. For the virulence tests evaluated in vitro, yeasts were grown in the presence and absence of 1000 g/mL of the extract. A ratio of 10: 1 (Candida spp. cells x HBECs) was incubated for 1 hour at 37 ° C, 200 rpm, fixed with 10% formalin and the number of Candida cells adhered to 150 HBEC determined by optical microscope. Biofilms were formed on polystyrene microplates in the presence or absence of the extract. The quantification was performed with crystal violet staining at 570 nm. All isolates were viable and exhibited phenotypic characteristics suggestive of each species identified. Two strains presumptively identified as Candida dubliniensis belonged to this species as determined with genotyping ABC, while strains identified as belonging to the Candida parapsilosis species complex were differentiated by RAPD genotyping. Candida albicans was found to be the most adherent species to the buccal epithelia, while C. tropicalis showed remarkable biofilm formation.We could detect that the extract of E. uniflora was able to reduce adhesion to HBEC for both Candida albicans and non-Candida albicans Candida species. On the other hand, only 16 Candida spp. strains (36 %) showed reduced biofilm formation. However, two highly biofilm producer strains of C. tropicalis had an expressive reduction in biofilm formation. This study reinforces the idea that besides growth inhibition, E. uniflora may interfere with the expression of some virulence factors of Candida spp., and may be possibly applied in the future as a novel antifungal agent.

Impacto na epidemiologia da sepse tardia após a mudança do protocolo de antimicrobianos em uma UTIN de um serviço universitário

Advances in neonatology resulted in reducing the mortality rate and the consequent increase in survival of newborn pre terms (PTN). On the other hand, there was also a considerable increase in the risk of developing health care-related infection (HAI) in its most invasive, especially for bloodstream. This situation is worrying, and prevent the occurrence of it is a challenge and becomes one of the priorities in the Neonatal Intensive Care Unit (NICU). Sepsis is the main cause of death in critical neonates and affects more than one million newborns each year, representing 40% of all deaths in neonates. The incidence of late sepsis can reach 50% in NICUs. Currently the major responsible for the occurrence of sepsis in developed countries is the coagulase negative Staphylococcus (CoNS), followed by S. aureus. The cases of HAIs caused by resistant isolates for major classes of antimicrobial agents have been increasingly frequent in the NICU. Therefore, vancomycin has to be prescribed more frequently, and, today, the first option in the treatment of bloodstream infections by resistant Staphylococcus. The objectives of this study were to assess the impact on late sepsis in epidemiology III NICU after the change of the use of antimicrobials protocol; check the frequency of multiresistant microorganisms; assess the number of neonates who came to death. This study was conducted in NICU Level III HC-UFU. three study groups were formed based on the use of the proposed late sepsis treatment protocol, with 216 belonging to the period A, 207 B and 209 to the C. The work was divided into three stages: Period A: data collected from neonates admitted to the unit between September 2010 to August 2011. was using treatment of late sepsis: with oxacillin and gentamicin, oxacillin and amikacin, oxacillin and cefotaxime. Period B: data were collected from March 2012 to February 2013. Data collection was started six months after protocol change. Due to the higher prevalence of CoNS, the initial protocol was changed to vancomycin and cefotaxime. Period C: data were collected from newborns inteerne in the unit from September 2013 to August 2014. Data collection was started six months after the protocol change, which occurred in March 2013. From the 632 neonates included in this study, 511 (80,8%) came from the gynecology and obstetrics department of the HC-UFU. The mean gestational age was 33 weeks and the prevailing sex was male (55,7%). Seventy-nine percent of the studied neonates were hospitalized at the NICU HC-UFU III because of complications related to the respiratory system. Suspicion of sepsis took to hospitalization in the unit of 1,9% of newborns. In general, the infection rate was 34,5%, and the most frequent infectious sepsis syndrome 81,2%. There was a tendency to reduce the number of neonates who died between periods A 11 and C (p = 0,053). From the 176 cases of late sepsis, 73 were clinical sepsis and 103 had laboratory confirmation, with greater representation of Gram positive bacteria, which corresponded to 67.2% of the isolates and CoNS the most frequent micro-organism (91,5%). There was a statistically significant difference in the reduction of isolation of Gram positive microorganisms between periods A and C (p = 0,0365) as well as in reducing multidrug-resistant CoNS (A and B period p = 0,0462 and A and C period, p = 0,158). This study concluded that: the CoNS was the main microorganism responsible for the occurrence of late sepsis in neonates in the NICU of HC-UFU; the main risk factors for the occurrence of late sepsis were: birth weight <1500 g, use of PICC and CUV, need for mechanical ventilation and parenteral nutrition, SNAPPE> 24 and length of stay more than seven days; the new empirical treatment protocol late sepsis, based on the use of vancomycin associated cefepime, it was effective, since promoted a reduction in insulation CoNS blood cultures between the pre and post implementation of the Protocol (A and C, respectively); just as there was a reduction in the number of newborns who evolved to death between periods A and C.

Desarrollo de nuevas formulaciones tópicas de anfotericina B para el tratamiento de micosis

Las infecciones por hongos se han convertido en un tema de gran preocupación en todo el mundo, se estima que más de 40 millones de personas sufren infecciones por hongos, tanto en países desarrollados como en países en vías de desarrollo (Güngör, et al., 2013). Las micosis superficiales se encuentran entre las formas más frecuentes de infecciones en los humanos. Se estima que afectan un 20-25 % de la población mundial y su incidencia está constantemente en aumento (Vena, et al., 2012) (Havlickova, et al., 2008) (Das, et al., 2007). Actualmente, este tipo de infecciones son un frecuente motivo de consulta para el médico de familia (Hernández, et al., 2014) y el dermatólogo. Lo cual nos obliga a permanecer constantemente actualizados La candidiasis es la micosis emergente con mayor efecto en el ser humano debido a su frecuencia y a la gravedad de sus complicaciones (López-Martínez, R., 2010). La candidiasis superficial es una de las formas clínicas más comunes. Es característicamente crónica y recurrente, y, a veces, indica el comienzo de las formas graves de esta micosis (Pappas, et al., 2009). Las levaduras del género Candida son microorganismos pertenecientes a la microbiota normal de individuos sanos, principalmente en la mucosa oral, el tracto gastrointestinal y el tracto genitourinario femenino (Shao, et al., 2007). Sin embargo, estos hongos son responsables de diferentes manifestaciones clínicas, especialmente en pacientes inmunocomprometidos, que van desde infecciones de la piel y mucosas a infecciones sistémicas (Sardi, et al., 2013). Su importancia viene de la alta frecuencia con que colonizan e infectan el huésped humano (De Bernardis, et al., 2004), siendo el cuarto patógeno más común asociado con los casos de infección nosocomial (Wisplinghoff, et al., 2004)...

International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus

Acknowledgements We thank Gilead Sciences Europe Ltd., UK for financially supporting logistical aspects of this study. The logistics of the meeting were handled by Congress Care, Den Bosch, The Netherlands. The sponsor was not involved in the selection of the participants or procedures, or in the discussion, data collection, analysis or writing of the manuscript. The medical writer was financially supported by the Dutch Society for Medical Mycology and the Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.

Association between vaginal bacterial microbiota and the presence and clinical presentation of vulvovaginal candidiasis

Thesis (Master's)--University of Washington, 2016-08

FUNCTIONAL CHARACTERIZATION OF THE LINK BETWEEN CARBOHYDRATE METABOLISM AND THE PATHOGENESIS OF THE INVASIVE M1T1 GROUP A STREPTOCOCCUS

The Group A Streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen that colonizes a variety of sites within the host. Infections can vary from minor and easily treatable, to life-threatening, invasive forms of disease. In order to adapt to niches, GAS utilizes environmental cues, such as carbohydrates, to coordinate the expression of virulence factors. Research efforts to date have focused on identifying how either components of the phosphoenolpyruvate-phosphotransferase system (PTS) or global transcriptional networks affect the regulation of virulence factors, but not the synergistic relationship between the two. The present study investigates the role of a putative PTS-fructose operon encoded by fruRBA and its role in virulence in the M1T1 strain 5448. Growth in fructose resulted in induction of fruRBA. RT-PCR showed that fruRBA formed an operon, which was repressed by FruR in the absence of fructose. Growth and carbon utilization profiles revealed that although the entire fruRBA operon was required for growth in fructose, FruA was the main fructose transporter. The ability of both ΔfruR and ΔfruB mutants to survive in whole human blood or neutrophils was impaired. However, the phenotypes were not reproduced in murine whole blood or in a mouse intraperitoneal infection, indicating a human-specific mechanism. While it is known that the PTS can affect activity of the Mga virulence regulator, further characterization of the mechanism by which sugars and its protein domains affect activity have not been studied. Transcriptional studies revealed that the core Mga regulon is activated more in a glucose-rich than a glucose-poor environment. This activation correlates with the differential phosphorylation of Mga at its PTS regulatory domains (PRDs). Using a 5448 mga mutant, transcriptome studies in THY or C media established that the Mga regulon reflects the media used. Interestingly, Mga regulates phage-encoded DNases in a low glucose environment. We also show that Mga activity is dependent on C-terminal amino acid interactions that aid in the formation of homodimers. Overall, the studies presented sought to define how external environmental cues, specifically carbohydrates, control complex regulatory networks used by GAS, contribute to pathogenesis, and aid in adaptation to various nutrient conditions encountered.