8 research outputs found
Analysis of meticillin-susceptible and meticillin-resistant biofilm-forming Staphylococcus aureus from catheter infections isolated in a large Italian hospital
Several characteristics were analysed in 37 Staphylococcus aureus isolates from nosocomial catheter infections: the PFGE profile after SmaI digestion of chromosomal DNA, the ability to form a biofilm on a polystyrene surface, antibiotic susceptibility patterns (penicillin, oxacillin, erythromycin, tetracycline, clindamycin, telithromycin, gentamicin, ciprofloxacin, quinupristin/dalfopristin, rifampicin, vancomycin and linezolid), and the presence of genetic determinants of antibiotic resistance and biofilm formation. All strains but three (92 %) were able to grow on a plastic surface as a biofilm. An almost complete association was found between phenotypes and genotypic traits of antibiotic resistance, whilst PFGE profiling showed the highly polyclonal composition of the set of strains under study. Sixteen isolates (43 %) were meticillin-resistant and were subjected to staphylococcal cassette chromosome mec (SCCmec) and cassette chromosome recombinase (ccr) complex type determination by multiplex PCR. Only a subgroup of six strains belonged to the archaic clone PFGE type and bore the SCCmec/ccrAB type I structure. Among the remaining strains some presented small rearrangements of the SCCmec/ccrAB genetic locus, whilst others could barely be traced back to a known structural type. These observations suggest that, at the local level and at a particular site of infection, S. aureus may show great genetic variability and escape the general rule of expansion of the S. aureus pandemic clones
The application of oligonucleotide probes and microarrays for the identification of freshwater diatoms.
Diatoms are major contributors to global
carbon fixation and constitute a significant portion of
biofilms found in lotic ecosystems. Despite their
widespread abundance and the fact that extensive
studies have been performed on morphological features
of frustules, molecular tools for the identification
of diatoms are not commonly available. This study
focuses on the development of oligonucleotide probes
for the detection of diatom species relevant to water
quality assessment. The selected panel of diatoms covers all the species found in water of varying quality
from the rivers of central-East Apennine (Italy). Small
subunit rRNA-targeted probes were applied to a
microarray platform as well as to a new technique
termed Primer–Probe, with the aim of obtaining a
molecular tool suitable for accurate identification of
both single and mixed species diatom populations. The
Primer–Probe technique together with dot-blot assays
proved to be ideal for the preliminary screening of a
large set of DNA oligonucleotides designed by ARB
software. It was shown that microarrays, as a promising
technology for rapid and simultaneous detection
of a wide range of species-specific genetic markers,
can be adapted to monitor changes within a diatom
community. It is suggested that microarrays will
provide a molecular basis for microbial identification
to support standard microscopy techniques used by
ecologists and environmental scientists for monitoring
water quality
Detection of phage-associated virulence/resistance genes in induced prophages of Streptococcus pyogenes clinical isolates
Objectives: The genome of each sequenced Streptococcus pyogenes strain shows to contain many prophages encoding proven or putative extracellular virulence factors and antibiotic resistance determinants. In this work we determined the presence of prophage-associated virulence/resistance genes (F-vir) and the induction profile of prophages harbouring virulence/resistance genes in fifty-nine S. pyogenes pharyngeal isolates.
Methods: PCR was used to assess the presence of those genes (i.e. speA, speC, speH, speI, speK, speL, speM, ssa, spd1, spd3, spd4, sdn, sda, sla, mefA and TetO) known to be prophage-associated in S. pyogenes. Each strain was treated with mitomycin C to induce release of functional phages, and the corresponding unrestricted total DNA was then analysed by pulsed field gel electrophoresis (PFGE). S. pyogenes SF370 and strain-6 (mefA/TetO) were used as control strains. Single or multiple bands, corresponding to phage DNA, were obtained only in mitomycin treated cells. They were excised and analysed by PCR to detect the specific F-vir.
Results: Five percent of the strains did not contain any of the known F-vir, while 76% had at least two. The distribution of F-vir was greatly variable and the overall mean number of F-vir per isolate was 3.8 (± 2.3). The release of phage DNA was achieved in 17 strains. Among these, PCR analysis detected a single F-vir in the phage DNA released by 35.3% of the strains, and three F-vir in 17.6%. One strain released phage DNA containing two F-vir, whereas another strain released phage DNA positive to five F-vir. The F-vir most frequently associated with released phage DNAs were sdn, spd4, speC, spd1 and spd3.
Conclusion: The pharynx is colonised by S. pyogenes harbouring a variable number and assortment of F-vir. A limited number of virulence genes are hosted by functional prophages and, therefore, have the potentiality to be horizontally transferred. Many strains possess different important F-vir that, at least in the conditions used, cannot be detected in released phage DNAs. These results suggest that the population of Fvir-lacking functional prophages is vast and that the polylysogeny would be the product of the accumulation of temperate phages that eventually become defective
IL-32γ potentiates tumor immunity in melanoma.
Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non-T cell-inflamed TME
IL-32γ potentiates tumor immunity in melanoma
Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non-T cell-inflamed TME
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IL-32γ potentiates tumor immunity in melanoma
Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non-T cell-inflamed TME