Retrovirology. 2012 Apr 1;9(1):29. [Epub ahead of print]
Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity.
van Maarseveen NM, Andersson D, Lepsik M, Fun A, Schipper PJ, de Jong D, Boucher CA, Nijhuis M.
BACKGROUND: Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V).
RESULTS: Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one. The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant.
CONCLUSIONS: The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.
PLoS One. 2012;7(3):e32589. Epub 2012 Mar 16.
Direct Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Improves Appropriateness of Antibiotic Treatment of Bacteremia.
Vlek AL, Bonten MJ, Boel CH.
Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01)). Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.
BMC Infect Dis. 2012 Mar 22;12(1):68. [Epub ahead of print]
Dynamics of ampicillin-resistant Enterococcus faecium clones colonizing hospitalized patients: Data from a prospective observational study.
Weisser M, Oostdijk EA, Willems RJ, Bonten MJ, Frei R, Elzi L, Halter J, Widmer AF, Top J.
BACKGROUND: Little is known about the dynamics of colonizing Enterococcus faecium clones during hospitalization, invasive infection and after discharge.
METHODS: In a prospective observational study we compared intestinal E. faecium colonization in three patient cohorts: 1) Patients from the Hematology Unit at the University Hospital Basel (UHBS), Switzerland, were investigated by weekly rectal swabs (RS) during hospitalization (group 1a, n = 33) and monthly after discharge (group 1b, n = 21). 2) Patients from the Intensive Care Unit (ICU) at the University Medical Center Utrecht, the Netherlands (group 2, n = 25) were swabbed weekly. 3) Patients with invasive E. faecium infection at UHBS were swabbed at the time of infection (group 3, n = 22). From each RS five colonies with typical E. faecium morphology were picked. Species identification was confirmed by PCR and ampicillin-resistant E. faecium (ARE) isolates were typed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). The Simpson's Index of Diversity (SID) was calculated.
RESULTS: Out of 558 ARE isolates from 354 RS, MT159 was the most prevalent clone (54%, 100%, 52% and 83% of ARE in groups 1a, 1b, 2 and 3, respectively). Among hematological inpatients 13 (40%) had ARE. During hospitalization, the SID of MLVA-typed ARE decreased from 0.745 [95%CI 0.657-0.833] in week 1 to 0.513 [95%CI 0.388-0.637] in week 3. After discharge the only detected ARE was MT159 in 3 patients. In the ICU (group 2) almost all patients (84%) were colonized with ARE. The SID increased significantly from 0.373 [95%CI 0.175-0.572] at week 1 to a maximum of 0.808 [95%CI 0.768-0.849] at week 3 due to acquisition of multiple ARE clones. All 16 patients with invasive ARE were colonized with the same MLVA clone (p < 0.001).
CONCLUSIONS: In hospitalized high-risk patients MT159 is the most frequent colonizer and cause of invasive E. faecium infections. During hospitalization, ASE are quickly replaced by ARE. Diversity of ARE increases on units with possible cross-transmission such as ICUs. After hospitalization ARE are lost with the exception of MT159. In invasive infections, the invasive clone is the predominant gut colonizer.
PLoS Pathog. 2012 Mar;8(3):e1002606. Epub 2012 Mar 22.
Inactivation of staphylococcal phenol soluble modulins by serum lipoprotein particles.
Surewaard BG, Nijland R, Spaan AN, Kruijtzer JA, de Haas CJ, van Strijp JA.
Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.
J Med Microbiol. 2012 Mar 1. [Epub ahead of print]
Detection of carbapenemase producing Enterobacteriaceae with a commercial DNA Microarray.
Cohen Stuart J, Voets G, Scharringa J, Fluit A, Leverstein-Van Hall M.
University Medical Centre Utrecht.
The Check-MDR CT102 DNA microarray enables detection of the most prevalent carbapenemases (NDM, VIM, KPC, OXA-48 and IMP) and ESBLs (SHV, TEM and CTX-M). The test performance of this microarray was evaluated with 95 Enterobacteriaceae isolates suspected for carbapenemase production, i.e. with meropenem MICs ≥0.5 mg/L. The isolate collection contained 70 carbapenemase producing isolates (37 KPC, 20 VIM, 5 OXA-48, 4 KPC plus VIM, 4 NDM), and 25 carbapenemase gene negative isolates. An ESBL was produced by 51 of the isolates. PCR and sequencing of beta-lactamase genes was used as reference test. For detection of carbapenemases, the sensitivity of the microarray was 97% (68/70), with 100% specificity. The two negative isolates tested positive when microarray test was repeated (an OXA-48 and a KPC producing isolate). For ESBL detection, the sensitivity was 100% (51/51) and the specificity 98% (43/44), although 20% of the SHV-12 ESBLs were categorized as SHV-2 like ESBLs. In conclusion, the CDT102 microarray is a rapid and accurate tool for detection of carbapenemase and ESBL genes, although the array seems less suitable for epidemiology of ESBL genes.
Crit Care. 2012 Feb 7;16(1):R22.
Costs and benefits of rapid screening of methicillin-resistant Staphylococcus aureus carriage in intensive care units: a prospective multicenter study.
Wassenberg M, Kluytmans J, Erdkamp S, Bosboom R, Buiting A, van Elzakker E, Melchers W, Thijsen S, Troelstra A, Vandenbroucke-Grauls C, Visser C, Voss A, Wolffs P, Wulf M, van Zwet T, de Wit A, Bonten M.
INTRODUCTION: Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is a cornerstone of successful MRSA control policies. Implementation of such strategies is hampered when using conventional cultures with diagnostic delays of 3 to 5 days, as many non-carriers remain unnecessarily isolated. Rapid diagnostic testing (RDT) reduces the amount of unnecessary isolation days, but costs and benefits have not been accurately determined in intensive care units (ICUs).
METHODS: Embedded in a multi-centre hospital-wide study in 12 Dutch hospitals we quantified cost per isolation day avoided using RDT for MRSA, added to conventional cultures, in ICUs. BD GeneOhmTM MRSA PCR (IDI) and Xpert MRSA (GeneXpert) were subsequently used during 17 and 14 months, and their test characteristics were calculated with conventional culture results as reference. We calculated the number of pre-emptive isolation days avoided and incremental costs of adding RDT.
RESULTS: 163 patients at risk for MRSA carriage were screened and MRSA prevalence was 3.1% (n=5). Duration of isolation was 27.6 and 21.4 hours with IDI and GeneXpert, respectively, and would have been 96.0 hours when based on conventional cultures. The negative predictive value was 100% for both tests. Numbers of isolation days were reduced by 44.3% with PCR-based screening at the additional costs of E327.84 (IDI) and E252.14 (GeneXpert) per patient screened. Costs per isolation day avoided were E136.04 (IDI) and E121.76 (GeneXpert).
CONCLUSIONS: In a low endemic setting for MRSA, RDT safely reduced the number of unnecessary isolation days on ICUs by 44%, at the costs of E121.76 to E136.04 per isolation day avoided.
AIDS. 2012 Feb 20;26(4):465-474.Long-term complications in patients with poor immunological recovery despite virological successful HAART in Dutch ATHENA cohort.
van Lelyveld SF, Gras L, Kesselring A, Zhang S, De Wolf F, Wensing AM, Hoepelman AI; on behalf of the ATHENA national observational cohort study.
Department of Internal Medicine & Infectious Diseases, University Medical Center Utrecht, Utrecht, Stichting HIV Monitoring, Amsterdam, Department of Virology, Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
OBJECTIVE: We investigated the risk of AIDS and serious non-AIDS-defining diseases (non-ADDs) according to the degree of immunological recovery after 2 years of virological successful antiretroviral therapy (HAART).
DESIGN: Retrospective observational cohort study including HIV-infected patients treated with HAART resulting in viral suppression (<500 copies/ml).
METHODS: Patients were grouped according to their CD4 cell count after 2 years of HAART: CD4 cell count less than 200 cells/μl (group A), 200-350 cells/μl (group B), 351-500 cells/μl (group C) or more than 500 cells/μl (group D). Analysis was done to assess predictors for poor immunological recovery and the occurrence of a composite endpoint [death, AIDS, malignancies, liver cirrhosis and cardiovascular events (CVEs)], non-ADDs, CVEs and non-AIDS-defining malignancies (non-ADMs).
RESULTS: Three thousand and sixty-eight patients were included. Older age, lower CD4 cell nadir and lower plasma HIV-RNA at the start of HAART were independent predictors for a poor immunological recovery. The composite endpoint, non-ADDs and CVE were observed most frequently in group A (overall log rank, P < 0.0001, P = 0.002 and P = 0.01). In adjusted analyses, age was a strong independent predictor for all endpoints. Compared with group A, patients in group D had a lower risk for the composite endpoint [hazard ratio 0.54 (95% confidence interval [CI] 0.33-0.87]; patients in group B had a lower risk for CVEs [hazard ratio 0.34 (95% CI 0.14-0.86)].
CONCLUSION: Poor immunological recovery despite virological successful HAART is associated with a higher risk for overall morbidity and mortality and CVEs in particular. This study underlines the importance of starting HAART at higher CD4 cell counts, particularly in older patients.
Cell Microbiol. 2012 Feb 6.
Identification of an immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa).
Bardoel BW, Hartsink D, Vughs MM, de Haas CJ, van Strijp JA, van Kessel KP.
Department of Medical Microbiology, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands.
Phagocytosis by neutrophils is the essential step in fighting Pseudomonas infections. The first step in neutrophil recruitment to the site infection is the interaction of P-selectin (on endothelial cells) with P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils. Pseudomonas aeruginosa secretes various proteases that degrade proteins that are essential for host defence, such as elastase and alkaline protease. Here we identify PA0572 of P. aeruginosa as an inhibitor of PSGL-1 and named this secreted hypothetical protease immunomodulating metalloprotease of P. aeruginosa or IMPa. Proteolytic activity was confirmed by cleavage of recombinant and cell-surface expressed PSGL-1. Functional inhibition was demonstrated by impaired PSGL-1-mediated rolling of IMPa-treated neutrophils under flow conditions. Next to PSGL-1, IMPa targets CD43 and CD44 that are also involved in leucocyte homing. These data indicate that IMPa prevents neutrophil extravasation and thereby protects P. aeruginosa from neutrophil attack.
J Innate Immun. 2012 Feb 7.Staphylococcus aureus Virulence Is Enhanced by Secreted Factors That Block Innate Immune Defenses.
Jongerius I, von Köckritz-Blickwede M, Horsburgh MJ, Ruyken M, Nizet V, Rooijakkers SH.
Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
Staphylococcus aureus is a leading human pathogen that causes a large variety of diseases. In vitro studies have shown that S. aureus secretes several small proteins that block specific elements of the host innate immune system, but their role in bacterial pathogenicity is unknown. For instance, the extracellular complement-binding protein (Ecb) impairs complement activation by binding to the C3d domain of C3. Its homolog, the extracellular fibrinogen-binding protein (Efb), is known to block both complement activation and neutrophil adhesion to fibrinogen. Here, we show that targeted inactivation of the genes encoding Ecb and Efb strongly attenuates S. aureus virulence in a murine infection model: mice experienced significantly higher mortality rates upon intravenous infection with wild-type bacteria (79%) than with an isogenic ΔEcbΔEfb mutant (21%). In addition, Ecb and Efb are both required for staphylococcal persistence in host tissues and abscess formation in the kidneys (27% for wild-type vs. 7% for the ΔEcbΔEfb mutant). During staphylococcal pneumonia, Ecb and Efb together promote bacterial survival in the lungs (p = 0.03) and block neutrophil influx into the lungs. Thus, Ecb and Efb are essential to S. aureus virulence in vivo and could be attractive targets in future vaccine development efforts.
J Virol. 2012 Jan;86(1):572-7. Epstein-Barr virus isolates retain their capacity to evade T cell immunity through BNLF2a despite extensive sequence variation.
Horst D, Burrows SR, Gatherer D, van Wilgenburg B, Bell MJ, Boer IG, Ressing ME, Wiertz EJ.
Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.
The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the Transporter associated with Antigen Processing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed in natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.