Induction of innate immune responses by KPC-producing Klebsiella pneumoniae of the pandemic sequence type 258-clade I.

Klebsiella pneumoniae -carbapenemase-producing K. pneumoniae (KPC) sequence-type 258 (ST258) has emerged as an important human pathogen throughout the world. Although lacking known virulence factors, it is associated with significant morbidity and high mortality rates. The pathogenicity of KPC K. pneumoniae ST258 strains has not been fully elucidated yet. We sought to investigate the interactions of the KPC K. pneumoniae ST258-clade I with different components of innate immunity. Human serum was used to evaluate the serum bactericidal activity and the J774A.1 murine (BALB/c mice) macrophage cell-line was used to examine phagocytosis, mRNA expression and production of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6. L-78, a KPC-producing K. pneumoniae ST258-clade I strain was used as representative of the strains circulating in Greek hospitals. K. pneumoniae ATCC 43816, a virulent K2 strain, was used for comparison. Strain L-78 was susceptible to human serum and rapidly phagocytosed by J774A.1 cells, in contrast to the virulent K2 strain, which was serum-resistant and slowly phagocytosed. Stimulation of the J774A.1 cells with the L-78 strain induced production of IL-1ß at concentration levels significantly higher compared to K2, whereas production of TNF-α and IL-6 levels were comparable by the two strains. L-78 was able to induce IL-1ß mRNA and NLRP3 mRNA expression. Our findings indicate that K. pneumoniae ST258-clade I is serum sensitive, rapidly phagocytosed and is capable of eliciting adequate innate immune response in terms of production of pro-inflammatory cytokines.

Changes in the rates and population structure of methicillin-resistant Staphylococcus aureus (MRSA) from bloodstream infections: A single-centre experience (2000-2015).

OBJECTIVES: The aim of this study was to assess the rate of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSIs) and the population structure of MRSA isolates recovered between 2000-2015 in a tertiary-care hospital in Athens, Greece. METHODS: Non-duplicate MRSA blood isolates recovered during the study period were examined. Antimicrobial susceptibility testing was performed by Kirby-Bauer and gradient strip methods. Carriage of PVL and mecA genes was examined by PCR. Genetic relatedness of the isolates was studied by SCCmec, spa and multilocus sequence typing. RESULTS: A total of 398 MRSA BSI cases were identified. A decreasing trend in incidence from 1.69/10 000 patient-days in 2000 to 1.39/10 000 patient-days in 2015 (P=0.038) and in prevalence from 64.7% to 36.4% (P=0.008), respectively, was observed, whereas the incidence of methicillin-susceptible S. aureus BSI increased. MRSA isolates exhibiting resistance to common antistaphylococcal agents (excluding glycopeptides and the newer antistaphylococcals) decreased from 84.8% in 2000 to 0% in 2011 and were progressively 'replaced' by more susceptible phenotypes. A strong association between antimicrobial resistance phenotype and molecular type was observed. The pandemic HA-MRSA clone ST239-III progressively declined in parallel with increasing isolation frequency of two clonal complexes (CCs): HA-MRSA CC5, with the majority of isolates belonging to ST5-II; and CA-MRSA CC80, represented mainly by ST80-IV-t044, PVL+. CONCLUSION: The decline in MRSA BSI rates observed in our institution was associated with changes in population structure of the organism. This decline may be related to biological properties of the prevailing MRSA clones.

Colistin resistance in carbapenemase-producing Klebsiella pneumoniae bloodstream isolates: Evolution over 15 years and temporal association with colistin use by time series analysis.

Colistin is often the only available treatment option against infections caused by carbapenemase-producing Klebsiella pneumoniae (CP-Kp). In this study, the evolution of colistin resistance among CP-Kp and its relationship with colistin use in a tertiary-care hospital in Athens, Greece, was investigated. All CP-Kp blood isolates recovered between January 2002 and June 2016 were tested for susceptibility to colistin by agar dilution and broth microdilution methods. Data on colistin use were collected from the pharmacy database. Time series of colistin use and resistance were analysed using the Box and Jenkins method. A transfer function model was built to quantify the dynamic relationship between colistin use and resistance. Overall, 313 CP-Kp isolates were identified. The percentage colistin resistance increased from 0% in 2002 to 26.9% in 2016 (R2 = 0.5, P < 0.01). A temporal association between colistin use and resistance was observed; an increase in colistin use by 1 DDD/100 patient-days led to a 0.05 increase in the incidence rate of colistin resistance. The time lag between the effect of colistin use on subsequent variations in colistin resistance was 3 months. Colistin use and prior levels of colistin resistance could explain 69% of colistin resistance; in the remaining 31%, other factors might have played a role. The results presented here demonstrate a significant temporal association between colistin use and colistin resistance. These findings have important implications in implementing strategies to contain colistin resistance.

PARP1-driven apoptosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is considered a malignancy resulting from defects in apoptosis. For this reason, targeting apoptotic pathways in CLL may be valuable for its management. Poly [ADP-ribose] polymerase 1 (PARP1) is the main member of a family of nuclear enzymes that act as DNA damage sensors. Through binding on DNA damaged structures, PARP1 recruits repair enzymes and serves as a survival factor, but if the damage is severe enough, its action may lead the cell to apoptosis through caspase activation, or necrosis. We measured the PARP1 mRNA and protein pretreatment levels in 26 patients with CLL and the corresponding posttreatment levels in 15 patients after 3 cycles of immunochemotherapy, as well as in 15 healthy blood donors. No difference was found between the pre- and posttreatment levels of PARP1, but we found a statistically significant relative increase of the 89 kDa fragment of PARP1 that is cleaved by caspases in the posttreatment samples, indicating PARP1-related apoptosis in CLL patients after treatment. Our findings constitute an important step in the field, especially in the era of PARP1 inhibitors, and may serve as a base for future clinical trials with these agents in CLL.

Survivin messenger RNA levels in Epstein-Barr virus-positive patients with leukemic low-grade B-cell lymphomas expressing the latent membrane protein 1: evidence of apoptotic function?

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. LMP1 expression has been detected in patients with chronic lymphocytic leukemia, but its properties have not been studied in patients with low-grade B-cell lymphomas. Recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B cells. We detected LMP1 messenger RNA (mRNA) in patients with leukemic low-grade B-cell lymphoma and correlated the expression of the antiapoptotic molecule survivin to that of LMP1 in this group of patients. PATIENTS AND METHODS: Peripheral whole blood from 64 patients with low-grade B-cell lymphoma was tested by quantitative reverse transcriptase-polymerase chain reaction (PCR) for the presence of the BXLF-1 gene of EBV, and positive samples were tested by conventional PCR for LMP1 expression. Accordingly, survivin mRNA levels were measured by quantitative reverse transcriptase PCR in all samples and compared between LMP1-positive (LMP1(+)) and LMP1(-) patients. RESULTS: The BXLF-1 gene was detected in 27 of 64 patients (42%). LMP1 was expressed in 22 of 27 (81%) EBV(+) patients. Survivin expression was found to be 6.36 times higher in LMP1(-) patients than in LMP1(+) patients (P = .008). CONCLUSION: Our results imply that in patients with non-EBV-related leukemic low-grade B-cell lymphoma, LMP1 expression is possibly correlated to apoptosis, as indicated by the lower survivin mRNA levels in LMP1(+) patients.

Evidence for regulation of oxidative stress by latent membrane protein 1 oncoprotein in patients with low-grade leukemic B cell lymphoma with latent Epstein-Barr virus infection.

The role of latent Epstein-Barr virus (EBV) infection in the pathogenesis of low-grade B cell non-Hodgkin lymphoma (B-NHL) has not been studied. We therefore investigated the incidence of latent EBV infection in a group of patients with leukemic low-grade B-NHL, as well as the incidence of viral latent membrane protein 1 (LMP1) oncoprotein expression in the same patient group. Furthermore, in an attempt to elucidate the role of this viral oncoprotein in non-EBV-related lymphomas, we correlated the expression of LMP1 with the level of oxidative stress, a parameter related to apoptosis. In the present study we detected lower levels of oxidative stress in the sera of LMP1-positive patients. This possibly implies an anti-apoptotic role of this viral oncoprotein in low-grade B cell lymphomas. However, LMP1 expression status did not affect expression of the major anti-apoptotic gene BCL-2.

Rituximab in the treatment of EBV-positive low grade B-cell lymphoma.

BACKGROUND: Following infection of B lymphocytes by Epstein Barr virus (EBV), the viral genome remains in the nucleus, and a latency phase is established, during which only a small proportion of the viral genes are expressed. Among them, LMP1 is essential for transformation. Rituximab is a potent agent used in the treatment of low grade B-cell lymphomas and is also widely used for the treatment of post-transplant lymphoproliferative disorders caused by EBV. The effect of rituximab treatment on the latent EBV infection in non-transplant patients with lymphoproliferative disorders has never been studied to our knowledge. PATIENTS AND METHODS: We studied, the effect of rituximab-based immunochemotherapy on the EBV status of 44 patients with leukemic low grade B-cell lymphoma. RESULTS: After three cycles of rituximab-based treatment, only 1/17 patients was still positive for EBV. DISCUSSION: Our results suggest that rituximab used in the treatment of EBV-positive low-grade lymphoma is efficient in eradicating the virus from the peripheral blood, a fact with potential implications in the course and prognosis of the disease.

Correlation of Fc-γ RIIA polymorphisms with latent Epstein-Barr virus infection and latent membrane protein 1 expression in patients with low grade B-cell lymphomas.

Fc-γ RIIA (CD32), a member of the family of Fc-γ receptors, participates in the phagocytosis of bound to antibody antigens. The effectiveness of this function varies for its several haplotypes, and it participates in the pathogenesis of viral infections, according to recent studies. The genetic locus of Fc-γ RIIA consists of two allelic genes: 131-Arg (R131) and 131-His (H131). Our aim was to correlate Fc-γ RIIA polymorphisms, by studying the prevalence of each allele using PCR-RFLPs (polymerase chain reaction-restriction fragment length polymorphisms), with latent Epstein-Barr virus (EBV) infection and the expression of latent membrane protein 1 (LMP1) in 40 patients with leukemic low grade B-cell lymphomas. R131 was found in 84.2% of EBV-positive patients, but only in 28.5% of EBV-negative patients (p = 0.001). A similar correlation was found for R131 and LMP1 expression (84.6% vs. 28.5%) (p = 0.002). Our results support the hypothesis that Fc-γ RIIA polymorphisms are a genetic risk factor for latent EBV infection and the expression of its oncogenic latency proteins.