[Show abstract][Hide abstract] ABSTRACT: Genotyping of Klebsiella pneumoniae is indispensable for management of nosocomial infections, monitoring of emerging strains -including extended-spectrum beta-lactamase (ESBL) producers-, and general epidemiology. Such objectives require a high-resolution genotyping method with a fixed scheme that allows (1) long-term retrospective and prospective assessment, (2) objective result readout and (3) library storage for database development and exchangeable results. We have developed a multiple-locus variable number tandem repeat analysis (MLVA) using a single-tube fluorescently primed multiplex PCR for 8 Variable Number Tandem Repeats (VNTRs) and automated fragment size analysis. The type allocation scheme was optimized using 224 K. pneumoniae clinical isolates, which yielded 101 MLVA types. The method was compared to the gold standard multilocus sequence typing (MLST) using a subset of these clinical isolates (n = 95) and found to be highly concordant, with at least as high a resolution but with considerably less hands-on time. Our results position this MLVA scheme as an appropriate, high-throughput and relatively low-cost tool for K. pneumoniae epidemiology.
PLoS ONE 01/2014; 9(3):e91209. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated the changes in antibiotic resistance from 1998 to 2009 of Klebsiella pneumoniae isolated from the intensive care units (ICUs) and urology services of 14 Dutch hospitals and the consequences for empirical therapy.
Quantitative antibiotic susceptibility testing of K. pneumoniae was performed in a central laboratory using a microbroth dilution method. Breakpoints were as defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The prevalence of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing isolates was determined.
A significant increase in resistance among ICU isolates was observed for ceftazidime (4.2%-10.8%), ciprofloxacin (5.8%-18.5%) and trimethoprim/sulfamethoxazole (11.9%-23.1%), and for cefuroxime (2.8%-7.9%) and trimethoprim/sulfamethoxazole (13.5%-27.8%) among urology isolates. Among ICU isolates the prevalence of ESBLs increased significantly from 2% to 8%. Carbapenemase production was not demonstrated. Among ICU isolates the prevalence of multidrug resistance increased and has been ≥12% since 2004. Among urology isolates multidrug resistance was highest in 2009 at 7.4%. Overall, resistance was significantly higher among ICU isolates.
We observed an increase in resistance among ICU and urology isolates and an increased prevalence of ESBLs among ICU isolates. Carbapenemase production was not demonstrated. A regular update of empirical treatment protocols based on actual surveillance data is justified.
Journal of Antimicrobial Chemotherapy 01/2011; 66(4):855-8. · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of rifampin as an adjunct in biofilm-associated infections is based on the ability to penetrate into biofilms and a presumed activity against dormant bacteria. Yet, its efficacy remains contradictory, and rifampin-resistant strains frequently emerge during therapy. Therefore, the efficacy against rifampin-susceptible and isogenic rifampin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) strains was evaluated. Biofilms were generated under static conditions using MSSA with various genetic backgrounds. Oxacillin alone or with rifampin at various concentrations was subsequently added, and after 24 h biomass and viable cell counts were determined. Upon rifampin addition, interstrain variations in viable count change, ranging from a tendency toward antagonism to synergy, were observed among all strains tested, irrespective of the genetic background of the strain. Similar variations were observed in changes in biomass. The decrease in viable count upon rifampin addition was negatively correlated to formation of large amounts of biomass, since strains embedded by more biomass showed a diminished reduction in viable count. Rifampin (1 microg/ml) as adjunct to oxacillin achieved greater reductions in biomass produced by most rifampin-susceptible isolates, ranging from 17 to 54%, compared to 4% for oxacillin alone. In contrast, rifampin had no additional value in reduction of biomass of isogenic rifampin-resistant mutants. At subinhibitory concentrations of rifampin (0.008 microg/ml), none of the strains tested yielded an extra reduction in biomass that was > or = 40%. In conclusion, the effects of rifampin as adjunct on biomass and viable count were unpredictable, and the use of rifampin against biofilm containing rifampin-resistant strains seems unwarranted.
Antimicrobial Agents and Chemotherapy 09/2010; 54(9):3907-12. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since it is unknown whether β-lactam antimicrobial agents can be used effectively against borderline oxacillin-resistant Staphylococcus aureus (BORSA) with oxacillin MICs ≥4 mg/L, the in vitro bactericidal activity and pharmacodynamic effect of oxacillin against clinical BORSA isolates was evaluated. Time-kill experiments with oxacillin were performed and the results compared with those obtained with vancomycin, daptomycin and linezolid against BORSA with oxacillin MICs ≥4 mg/L and BORSA with oxacillin MICs ≤2 mg/L. Furthermore, the effect of β-lactamase production and plasmid profile analysis were taken into account to clarify responses to oxacillin. Oxacillin killing activity was attenuated against BORSA compared with ATCC 29213 since the pharmacodynamic parameters revealed that the potency of oxacillin was markedly reduced (c. ten-fold) against BORSA with oxacillin MICs ≥4 mg/L. pBORa53-like plasmid-containing BORSA with oxacillin MICs ≤2 mg/L showed markedly more regrowth. In conclusion, oxacillin was non-effective in the eradication of either (i) BORSA with oxacillin MICs ≥4 mg/L or (ii) β-lactamase-hyperproducing BORSA (MICs ≤2 mg/L). Further investigation into β-lactam dosing strategies against different BORSA strains is warranted in order to avoid possible therapy failure.
Clinical Microbiology and Infection 07/2010; 16(7):979-85. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spa typing/based upon repeat pattern (BURP) sometimes cannot differentiate multilocus sequence typing (MLST) clonal complexes (CCs) within spa-CCs. It has been observed previously that virulence factors, such as collagen adhesin (CNA) and toxic shock syndrome toxin 1 (TSST-1), are associated with certain Staphylococcus aureus lineages. Analysis of methicillin-sensitive and methicillin-resistant S. aureus by spa typing/BURP and detection of CNA and TSST-1 observed an association between CNA and MLST CC1, 12, 22, 30, 45, 51, and 239 and between TSST-1 and MLST CC30. In spa-CC 012, associated with MLST CC7, CC15, and CC30, MLST CC30 could be distinguished from MLST CC7 and CC15 with CNA and TSST-1 as lineage-specific markers. Lineage-specific markers can overcome clustering of nonrelated MLST CCs into 1 spa-CC.
[Show abstract][Hide abstract] ABSTRACT: The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.
Journal of clinical microbiology 10/2009; 47(12):4090-5. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the usefulness of flucloxacillin as empirical therapy for putative Staphylococcus aureus infections in intensive care unit (ICU) patients in the Netherlands, the antibiotic resistance of S. aureus isolates from ICUs over a 13 year period was investigated.
From 1996 to 2008, 1146 consecutive S. aureus isolates from ICU patients in 14 large referral hospitals were collected. The susceptibility to relevant antibiotics was determined by microbroth dilution according to CLSI guidelines.
Resistance to flucloxacillin was only found in 12 isolates (1%). The resistance to clarithromycin, ciprofloxacin and moxifloxacin showed a significant trend over time, from 4.2% to 10.3%, from 1.0% to approximately 10% and from 0.0% to approximately 5.0%, respectively (P < 0.05). The resistance to penicillin, clindamycin and doxycycline increased over time, from 74% to 75%, from approximately 3.0% in 1996 to 3.2% in 2008 and from 2.2% in 1996 to 8.2% in 2008, respectively (P > 0.05). Resistance to cephalosporins, carbapenems, rifampicin and gentamicin was sporadically observed. No resistance was found to vancomycin, teicoplanin and linezolid.
The empirical choice of flucloxacillin in the case of putative S. aureus infections in patients admitted to ICUs in the Netherlands is still justified.
Journal of Antimicrobial Chemotherapy 09/2009; 64(5):1029-34. · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For us to assess the spread of methicillin-resistant Staphylococcus aureus (MRSA), typing of the staphylococcal cassette chromosome mec (SCCmec) is a valuable addition to existing typing methods, such as multilocus sequence typing (MLST). Traditional SCCmec typing assays, that is, that of Oliveira et al. and Ito et al., are polymerase chain reaction (PCR) based, requiring electrophoresis. We introduce a rapid, 2-well, multiplex real-time PCR assay that can be used directly on bacterial suspensions and is able to characterize SCCmec type I to V based on the detection of the ccr genes and the mec complex. The assay was evaluated on 212 clinical MRSA isolates from various countries, associated with MLST clonal complexes (CC) 1, 5, 8, 22, 30, and 45, as well as pig-associated CC398. When comparing the real-time PCR assay with traditional methods, the correct SCCmec element was identified in 209 (99%) of the 212 MRSA isolates. The new assay enables high-throughput analyses for SCCmec on large strain collections.
[Show abstract][Hide abstract] ABSTRACT: The characterization of 62 community-associated methicillin-sensitive Staphylococcus aureus (MSSA) isolates from 440 individuals in the Yogyakarta area of Indonesia in 2006 showed that: (i) almost half of the isolates were associated with methicillin-resistant S. aureus lineages [clonal complex (CC)1, CC8 and CC45] and (ii) ten Panton-Valentine leukocidin-positive isolates were associated with CC1 (n = 7), CC30 (n = 1) and CC51 (n = 2). The high Panton-Valentine leukocidin prevalence (16%) among S. aureus is of concern because these strains can cause severe infections and the introduction of staphylococcal cassette chromosome mec into virulent and epidemic MSSA could pose a serious public health threat.
Clinical Microbiology and Infection 07/2009; 16(1):92-4. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) differs among the 3 countries forming the Euregio Meuse-Rhin (EMR) region (Belgium, Germany, and the Netherlands), cross-border healthcare requires information about the spread of MRSA in the EMR. We investigated the emergence, dissemination, and diversity of MRSA clones in the EMR by using several typing methods. MRSA associated with clonal complexes 5, 8, 30, and 45 was disseminated throughout the EMR. Dutch isolates, mainly associated with sequence types (ST) ST5-MRSA-II, ST5-MRSA-IV, ST8-MRSA-IV, and ST45-MSRA-IV had a more diverse genetic background than the isolates from Belgium and Germany, associated with ST45-MRSA-IV and ST5-MRSA-II, respectively. MRSA associated with pigs (ST398-MRSA-IV/V) was found in the Dutch area of the EMR. Five percent of the MRSA isolates harbored Panton-Valentine leukocidin and were classified as community-associated MRSA associated with ST1, 8, 30, 80, and 89.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the methicillin-resistant Staphylococcus aureus (MRSA) clones isolated in a Dutch university hospital, situated near the borders of Belgium and Germany, between 2002 and 2006. MRSA strains (n = 175) were characterized using spa and SCCmec typing. The presence of Panton Valentine leukocidin (PVL) was determined. Between 2002 and 2005, ST5-MRSA-IV was predominant, and the spa type of ST5-MRSA-IV changed from t002 to t447. ST5-MRSA-I, ST5-MRSA-II, ST228-MRSA-I, and ST247-MRSA-I were also observed in this period. From 2004, the MRSA genetic background became more diverse, and in 2006, ST5-MRSA-IV was only sporadically observed. From 2005, ST5-MRSA-II, ST8-MRSA-IV, ST22-MRSA-IV, and ST45-MRSA-IV were increasingly observed. Several other MRSA clones, such as ST239-MRSA-III, were found sporadically. Four PVL-positive MRSA isolates were observed, associated with ST80-MRSA-IV and ST8-MRSA-IV. ST5-MRSA-I, ST5-MRSA-II, ST5-MRSA-IV, and ST228-MRSA-I have not been described previously in The Netherlands.
European Journal of Clinical Microbiology 02/2009; 28(6):631-9. · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since bacteria embedded in biofilms are far more difficult to eradicate than planktonic infections, it would be useful to know whether certain Staphylococcus aureus lineages are especially involved in strong biofilm formation. For this reason, in vitro biofilm formation of 228 clinical S. aureus isolates of distinct clonal lineages was investigated.
At 0.1% glucose, more than 60% of the S. aureus strains associated with multilocus sequence typing (MLST) clonal complex (CC)8 produced large amounts of biomass, compared to 0-7% for various other clonal lineages. Additionally, S. aureus bloodstream isolates associated with MLST CC8 and CC7 had similar biofilm forming capacities as their commensal counterparts. Furthermore, strong biofilm formation could not be attributed to a specific accessory gene regulator (agr) genotype, as suggested previously. The agr genotypes were strictly associated with the clonal lineages. Moreover, strong biofilm formation was not related to slime formation. Congo red agar (CRA) screening is therefore not useful as a qualitative screening method for biofilm formation.
The adherence to polystyrene surfaces under physiologic glucose concentration (0.1%) was dependent on the clonal lineage. Strains associated with MLST CC8 were markedly more often classified as strong biofilm former at glucose concentrations of 0%, 0.1% and 0.25%. The present study reveals that the MLST CC8 associated genetic background was a predisposing factor for strong biofilm formation in vitro, under all tested glucose concentrations.
[Show abstract][Hide abstract] ABSTRACT: We observed that, between 1999 and 2006, up to 50% of the methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream isolates in our hospital had a genetic background common to endemic methicillin-resistant S. aureus clones (clonal complex 5 [CC5], CC8, CC22, CC30, and CC45). Furthermore, several successful MSSA lineages, such as CC7 and CC15, were observed.
Journal of clinical microbiology 08/2008; 46(7):2438-41. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CMVs carry several genes that are homologous to genes of the host organism. These include genes homologous to those encoding chemokines (CKs) and G protein-coupled receptors (GPCRs). It is generally assumed that these CMV genes were hijacked from the host genome during the long co-evolution of virus and host. In light of the important function of the CK and GPCR families in the normal physiology of the host, it has previously been hypothesized that the CMV homologs of these proteins, CMV vCKs and vGPCRs, may also have a significant impact on this physiology, such that lifelong maintenance and/or replication of the virus within the infected host is guaranteed. In addition, several of these homologs were reported to have a major impact in the pathogenesis of infection. In this review, the current state of knowledge on the CMV vCKs and vGPCRs will be discussed.
Current topics in microbiology and immunology 01/2008; 325:221-42. · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The generation of antibodies against G protein-coupled receptors (GPCRs) can be technically challenging. A modified DNA immunization protocol was employed in order to generate polyclonal antibodies against two herpes virus-encoded GPCRs, i.e. Epstein-Barr virus (EBV) pBILF1 and rat cytomegalovirus (RCMV) pR78.
pBILF1 and pR78 expression plasmids were first injected into the tibialis anterior muscle of rats and rabbits, respectively. Subsequently, the uptake of plasmids by the muscle cells was facilitated through in vivo electroporation.
Potent antisera against both vGPCRs were obtained, as determined by immunoblot analysis and immunofluorescence. By using the antisera, we were able to show that the EBV BILF1 protein is expressed as a 45-kD, glycosylated protein, and that it is localized in the cytoplasmic membrane of EBV-infected cells. Interestingly, we found the R78-encoded vGPCRs to have unusual perinuclear localization in both R78-transfected and RCMV-infected cells.
The in vivo DNA electroporation method is a useful technique for generating antibodies against GPCRs.
Journal of pharmacological and toxicological methods 01/2008; 58(1):27-31. · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.
[Show abstract][Hide abstract] ABSTRACT: Cytomegalovirus (CMV) infection accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in both human and animal solid organ transplantation models. The host/viral mechanisms involved in this process are unclear. We examine the role of the rat CMV (RCMV)-encoded chemokine-receptor R33 in the development of TVS using a rat heart transplantation/CR model. F344 heart grafts were transplanted heterotopically into Lewis recipients. The ability of RCMV lacking the R33 gene (RCMV-Deltar33) to accelerate CR/TVS (neointimal index, NI) was compared to wild-type (WT) RCMV. Allograft recipients were infected with 1 x 10(5) pfu RCMV or RCMV-Deltar33 on postoperative day (POD) 1. Grafts from RCMV-Deltar33-infected recipients demonstrated an accelerated time to allograft CR compared to grafts from uninfected recipients (POD = 56 vs. 90), this was slower than that seen in grafts from WT-RCMV-infected recipients (POD = 45). Similarly, the degree of graft TVS formation at terminal rejection in RMCV-Deltar33 infected recipients was more severe than uninfected recipients (NI = 63 vs. 45), yet not as severe as in WT-RCMV infected recipients (NI = 83). In parallel, RCMV-Deltar33 failed to induce vascular smooth muscle cell (SMC) migration in vitro, whereas WT-RCMV induced substantial migration. The RCMV-encoded chemokine-receptor r33 is critical for RCMV-accelerated TVS/CR and vascular SMC migration.
American Journal of Transplantation 04/2005; 5(3):436-42. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesviruses. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has hitherto escaped attention, yet BILF1-like genes are conserved among all known lymphocryptovirus species, suggesting that they play a pivotal role in viral infection. To determine the function of EBV BILF1, the activity of this gene and its products was studied. BILF1-specific mRNA was detected in various EBV-positive cell types and found to be expressed predominantly during the immediate early and early phases of infection in vitro. Interestingly, in COS-7 cells transfected with BILF1 expression constructs, a decrease in forskolin-induced CRE-mediated transcription was measured, as well as an increase in NF-kappaB-mediated transcription. In contrast, CRE-mediated transcription was increased in EBV-positive Burkitt's lymphoma cells as well as EBV-positive lymphoblastoid B cells transfected with BILF1, whereas NF-kappaB-mediated transcription levels remained unaffected in these cells. All observed activities were sensitive to treatment with pertussis toxin, indicating that the BILF1-encoded protein mediates these activities by coupling to G proteins of the G(i/o) class. Finally, reduced levels of phosphorylated RNA-dependent antiviral protein kinase were observed in COS-7 and Burkitt's lymphoma cells transfected with BILF1. Neither of the observed effects required a ligand to interact with the BILF1 gene product, suggesting that BILF1 encodes a constitutively active GPCR capable of modulating various intracellular signaling pathways.
Journal of Virology 02/2005; 79(1):441-9. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rat cytomegalovirus (RCMV) possesses two adjacent genes, r131 and r129, which have the potential to encode CC chemokine homologs. Interestingly, the amino acid sequences encoded by both genes show similarity to the sequence of the murine CMV (MCMV) MCK-2 protein, which is encoded by the m131/129 gene. In order to study the significance of the r131 gene in the pathogenesis of RCMV infection, we generated two different virus strains in which the r131 open reading frame is disrupted. Replication of these null mutant strains, designated RCMVdeltar131a and RCMVdeltar131b, was evaluated in vitro and in vivo. Both strains were found to replicate with a similar efficiency as wild-type (WT) RCMV in vitro. However, in contrast to WT virus, neither RCMVdeltar131a nor RCMVdeltar131b established a high-titer infection in the salivary glands of immunocompromised rats. Furthermore, in a local, rat footpad infection model, both recombinant viruses induced a significantly lower amount of paw swelling than did WT RCMV. Also, a higher number of infiltrating macrophages was observed in paws infected with WT RCMV than in those infected with the recombinants. Taken together, these results suggest that r131 (i) promotes inflammation at initial sites of inoculation and, subsequently, efficient virus dissemination to or infection of the salivary glands and (ii) might be involved in the persistence of virus infection, at least in the spleen. In addition, our data indicate that r131 represents the functional homolog of the MCMV m131/129 gene.