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Objectives To evaluate a rapid diagnostic algorithm based on MALDI-TOF MS, lateral flow immunoassays (LFIAs) and molecular testing performed directly from positive blood cultures (BCs) for Gram-negative species identification and detection of CTX-M extended-spectrum β-lactamases and main carbapenemases. Methods Non-duplicate BCs positive to Gram-negative bacteria at microscope examination were subjected to species identification by direct MALDI-TOF MS following recovery of bacterial pellet by Rapid MBT Sepsityper® kit. Subsequently, NG-Test® CARBA 5 and NG-Test® CTX-M MULTI LFIAs were performed according to identified microbial species. Eazyplex® SuperBug CRE molecular assay was performed in cases of NG-Test® CARBA 5 negative results in patients with documented carbapenemase-producers carriage. Results of rapid diagnostic workflow were compared with those obtained by conventional diagnostic routine. Results Overall, the direct MALDI-TOF MS protocol allowed reliable identification to the species level of 92.1% of the 2133 monomicrobial BCs. Rate of matched identification was significantly higher for Enterobacterales (97.3%) in comparison to non-fermenting Gram-negative species (80.2%), obligate anaerobic bacteria (42.1%) and fastidious Gram-negative species (41.5%). The overall sensitivity of NG-Test® CARBA 5 and NG-Test® CTX-M MULTI was 92.2% and 91.6%, respectively. Integration of Easyplex® SuperBug CRE allowed the detection of blaKPC mutants associated with ceftazidime/avibactam resistance, reaching 100% sensitivity in carbapenemase detection. Both LFIAs and molecular testing showed no false-positive results. Conclusions Algorithms based on MALDI-TOF MS, LFIAs and molecular testing may represent a cost-effective tool to timely identify Gram-negative species and detect resistance markers directly from BCs. According to local epidemiology, these results may allow antimicrobial stewardship interventions including prompt use of new approved drugs.

Resistance to clinical triazoles in Aspergillus fumigatus is a growing concern for individuals at high risk of Aspergillus infection. Two triazole resistance selection routes are currently being investigated: one occurring in triazole-treated patients in healthcare settings, and the second taking place in the environment due to the widespread use of agricultural triazoles. This study aimed to assess the ability of agricultural azoles to promote cross-resistance to clinical azoles in A. fumigatus. Five A. fumigatus isolates susceptible to clinical azoles were exposed to the triazole 14α-demethylase inhibitor, tebuconazole (TBC), and then antifungal susceptibility tests for voriconazole, itraconazole, posaconazole and isavuconazole were performed. Under TBC selection pressure, all A. fumigatus isolates exhibited resistance to clinical triazoles. However, only two displayed a multiresistant phenotype to clinical azoles. TBC exposure was also associated with delayed conidia formation and progressive absence of conidiation. Noteworthy, no TBC-exposed clones harbored TR34/L98H mutation, as judged by real-time PCR assays. The observation that TBC exposure promotes cross-resistance to clinical triazoles warrants careful and thorough assessment of the human health risk associated with agricultural azoles. The absence of TR34/L98H mutation in cross-resistant A. fumigatus isolates suggests that other cyp51A mutations may be involved in clinical azole cross-resistance.

Narcisa Mandras,1 Monica Argenziano,2 Mauro Prato,1 Janira Roana,1 Anna Luganini,3 Valeria Allizond,1 Vivian Tullio,1 Nicole Finesso,4 Sara Comini,1 Bruno Emilio Bressan,4 Francesca Pecoraro,4 Giuliana Giribaldi,4 Adriano Troia,5 Roberta Cavalli,2 Anna Maria Cuffini,1,* Giuliana Banche1,* 1Department of Public Health and Pediatric Sciences, University of Torino, Turin, 10126, Italy; 2Department of Drug Science and Technology, University of Torino, Turin, 10125, Italy; 3Department of Life Sciences and Systems Biology, University of Torino, Turin, 10123, Italy; 4Department of Oncology, University of Torino, Turin, 10126, Italy; 5Istituto Nazionale di Ricerca Metrologica, Turin, 10135, Italy*These authors contributed equally to this workCorrespondence: Valeria Allizond, Department of Public Health and Pediatric Sciences, University of Torino, Via Santena 9, Turin, 10126, Italy, Tel +390116705644, Fax +390112365644, Email valeria.allizond@unito.itPurpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) – methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata – and for their physical interaction with cell walls/membranes.Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy.Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h.Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.Keywords: chitosan nanodroplets, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Candida spp, chronic wounds

Persistent hypoxia is a main clinical feature of chronic wounds. Intriguingly, oxygen-loaded nanodroplets (OLNDs), filled with oxygen-solving 2H,3H-decafluoropentane and shelled with polysaccharides, have been proposed as a promising tool to counteract hypoxia by releasing a clinically relevant oxygen amount in a time-sustained manner. Here, four different types of chitosan (low or medium weight (LW or MW), glycol-(G-), and methylglycol-(MG-) chitosan) were compared as candidate biopolymers for shell manufacturing. The aim of the work was to design OLND formulations with optimized physico-chemical characteristics, efficacy in oxygen release, and biocompatibility. All OLND formulations displayed spherical morphology, cationic surfaces, ≤500 nm diameters (with LW chitosan-shelled OLNDs being the smallest), high stability, good oxygen encapsulation efficiency, and prolonged oxygen release kinetics. Upon cellular internalization, LW, MW, and G-chitosan-shelled nanodroplets did not significantly affect the viability, health, or metabolic activity of human keratinocytes (HaCaT cell line). On the contrary, MG-chitosan-shelled nanodroplets showed very poor biocompatibility. Combining the physico-chemical and the biological results obtained, LW chitosan emerges as the best candidate biopolymer for future OLND application as a skin device to treat chronic wounds.

Prosthetic joint infection diagnosis is often difficult since biofilm-embedded microorganisms attach well to the prosthetic surfaces and resist their detection by conventional methods. DL-dithiothreitol has been described as a valid method for biofilm detachment on orthopedic devices. We report the case of an occasional detection of Listeria monocytogenes in a non immuno-compromised patient with a preoperative diagnosis of aseptic loosening. The infection diagnosis due to such rare bacteria was made postoperatively, thanks to a DL-dithiothreitol-based device. This may be considered a feasible approach for the microbiological analysis of prosthetic joint infection, considering that a prompt diagnosis of such biofilm-associated infections could bring some advantages, such as an early and appropriate antibiotic therapy administration and a reduction of undiagnosed infections.

Despite ongoing global efforts, antimicrobial resistance continues to threaten the treatment of an ever-increasing range of bacterial infections. There is substantial evidence that public education programs that foster microbial literacy amongst young school audiences may improve correct knowledge of specific health issues, such as prevention of microbial infections and responsible use of antibiotics. The aim of the Microbiological@mind project was to engage primary school students with the subject of microbiology, to promote both scientific interest and awareness towards correct behaviors that may ensure a safer lifestyle. Interactive workshops based on a full ''hands-on'' approach were carried out by an expert team from the University of Turin to over 1200 children aged 9-11 years at primary schools in Turin. A questionnaire (pre- and post-activity test) on the main topic (i.e. antibiotics) was used to assess project effectiveness. The workshops provided a useful means to strengthen the understanding of basic microbiology concepts amongst students. Students' baseline knowledge of antibiotics was quite low, as low percentages of correct answers on antibiotic action and use (5.0% and 12.1%, respectively) were found in the pre-activity tests. A significant increase (P

Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae, usually characterized by self-limiting regional lymphadenopathy and fever. Given the low clinical diagnostic sensitivity and specificity of conventional anti-B. henselae indirect immunofluorescence assays (IFAs), real-time polymerase chain reaction (PCR)-based detection of B. henselae is now being proposed as a more sensitive tool to diagnose CSD. Thus, here we have assessed the efficacy of real-time PCR in detecting B. henselae in different specimens from patients with suspected CSD and compared it to that of IFA. From March 2011 to May 2016, at the Microbiology and Virology Unit, Azienda Ospedaliera Universitaria Citta della Salute e della Scienza di Torino, Turin, Italy, 115 clinical specimens (56 aspirated pus, 39 fresh lymph node biopsies, and 20 whole blood samples) and 99 sera from 115 patients with suspected CSD (62 females and 53 males between the ages of 3 months and 68 years) were analyzed by both real-time PCR, used in a qualitative way, and IFA (IgM and IgG) for the presence of B. henselae. For 16 patients, serological results were not available due to a clinical decision not to request the test. B. henselae DNA positivity was detected by real-time PCR in 37.39% of patients, while 62.61% of them were negative. Thus, patients were divided into two groups: real-time PCR+ (n = 43) and real-time PCR- (n = 72). Real-time PCR screening of whole blood, biopsies, and aspirated pus revealed B. henselae positivity in 40%, 38.46%, and 35.71% of patients, respectively. When we analyzed samples by IFA, we found the presence of B. henselae in 28 out of 99 (28.28%) patients, of which 11 (11.11%) belonged to the real-time PCR+ group and 17 (17.17%) to the real-time PCR- group. Among the 71 seronegative subjects, 16 (16.16%) were found positive for B. henselae by real-time PCR. Thus, by combining the results of both assays, we were able to increase the percentage of B. henselae positive specimens from 27.27% (real-time PCR) or 28.28% (IFA) to 44.44% (real-time PCR+IFA). Altogether, these findings indicate that the early detection of B. henselae in patients with suspicious CSD through combined real-time PCR and serological analyses can lead to a more accurate diagnosis of CSD, thereby allowing prompt and appropriate disease management.

Lactobacilli have the potential to act as reservoirs of antibiotic resistance genes similar to those found in human pathogens, with the risk of transferring these genes to many pathogenic bacteria. In this study, we investigated the role of human polymorphonuclear cells (PMNs) against Lactobacillus spp. both resistant and susceptible to ciprofloxacin (a fluoroquinolone) and the effect of ciprofloxacin on the interaction between PMNs and three Lactobacillus spp. with different patterns of susceptibility to this drug. Hence, the primary functions of PMNs, such as phagocytosis and bacterial intracellular killing, against lactobacilli were investigated. The rate of PMN phagocytosis was high for ciprofloxacin-sensitive and ciprofloxacin-resistant strains. The patterns of intracellular killing of ciprofloxacin-sensitive and ciprofloxacin-resistant strains by PMNs underline that PMNs alone were able to kill lactobacilli. The addition of ciprofloxacin to PMNs did not result in a significant increase in the bacterial uptake by phagocytes. On the contrary, ciprofloxacin had a marked effect on PMN intracellular killing, resulting in increased numbers of killed ciprofloxacin-sensitive bacteria in comparison with antibiotic-free controls. Our data show that by itself, the profile of antibiotic resistance does not constitute an intrinsic factor of greater or lesser pathogenicity toward the host. The ability of PMNs to kill a diverse array of bacterial pathogens is essential for human innate host defense, primarily in immunocompromised patients.

Background The rate of acute infection after surgery for proximal humeral fractures is not known. The aims of this study were to report the incidence and to analyze the risk factors for infection after proximal humeral fracture treatment. Materials and methods We report a retrospective multicenter study of 452 proximal humeral fractures. Data were modeled by use of univariate and/or linear regression analyses to determine the odds ratio (OR). A logistic regression analysis was used to check for demographic and other characteristics with the potential to confound a true association between risk factors and infection. Results The mean age was 62.1 years, and 314 patients were female patients. Of the patients, 18 (4%) had an acute infection. The factors that correlated with infection were length of surgery (OR, 1.009; P = .05), preoperative skin preparation with chlorhexidine gluconate (OR, 0.13; P = .008), and prophylactic antibiotic (OR, 10.73; P = .03). The delay to surgery was close to achieving significance (OR, 1.71; P = .06). Conclusion This study suggests that washing the shoulder with chlorhexidine gluconate and avoiding the use of first-generation cephalosporin in favor of more effective prophylactic therapy are effective at reducing the risk of infection after treatment for proximal humeral fractures.

Aim: We evaluated the potential impact of caspofungin (CAS) on the functional activities of polymorphonuclear leukocytes (PMNs) from hemodialyzed patients (HDs) and renal transplant recipients (RTRs) against a multidrug-resistant clinical strain of Candida glabrata compared with those of PMNs from healthy subjects (HSs). Materials & methods: Effects of CAS on PMN phagocytosis and intracellular killing towards multidrug-resistant C. glabrata were evaluated in 66 HDs, 54 RTRs and 30 HSs in the absence and presence of CAS at MIC and sub-MICs. Results: When HD PMNs and RTR PMNs were exposed to both MICs and sub-MICs of CAS, their fungicidal activity against the multidrug-resistant C. glabrata strain was significantly higher than that of drug-free controls, with survival index values that overlapped with those achieved by HS PMNs. Conclusion: The obtained results underline the role of CAS in the restoration of the impaired PMN functions in HDs and RTRs. CAS might still constitute an effective therapeutic option for the treatment of invasive fungal infections caused by multidrug-resistant C. glabrata in patients with altered phagocyte-dependent innate immunity.

Vancomycin (Vm) currently represents the gold standard against methicillin-resistant Staphylococcus aureus (MRSA) infections. However, it is associated with low oral bioavailability, formulation stability issues, and severe side effects upon systemic administration. These drawbacks could be overcome by Vm topical administration if properly encapsulated in a nanocarrier. Intriguingly, nanobubbles (NBs) are responsive to physical external stimuli such as ultrasound (US), promoting drug delivery. In this work, perfluoropentane (PFP)-cored NBs were loaded with Vm by coupling to the outer dextran sulfate shell. Vm-loaded NBs (VmLNBs) displayed ∼300nm sizes, anionic surfaces and good drug encapsulation efficiency. In vitro, VmLNBs showed prolonged drug release kinetics, not accompanied by cytotoxicity on human keratinocytes. Interestingly, VmLNBs were generally more effective than Vm alone in MRSA killing, with VmLNB antibacterial activity being more sustained over time as a result of prolonged drug release profile. Besides, VmLNBs were not internalized by staphylococci, opposite to Vm solution. Further US association promoted drug delivery from VmLNBs through an in vitro model of porcine skin. Taken together, these results support the hypothesis that proper Vm encapsulation in US-responsive NBs might be a promising strategy for the topical treatment of MRSA wound infections.

The purpose of this investigation was to evaluate intracanal bacterial reduction by cryotreatment using a dental instrument equipped with a duct and connected to a cryogenic fluid source. A total of 86 roots were infected with Enterococcus faecalis and incubated. After incubation, the contaminated roots were divided into three study groups: 35 roots irrigated with 2 ml of a 5% sodium hypochlorite (NaOCl) solution, 35 roots irrigated with 2 ml of a 5% NaOCl solution and further treated with cryo and 10 roots irrigated with 2 ml of saline solution, plus positive and negative controls. Subsequent to each irrigation treatment, the residual bacterial colonies were counted. The use of cryo-instrumentation in association with NaOCl irrigation significantly reduced the number of Ent. faecalis (P < 0·01) in the root canal compared with controls. The interesting potential of cryotreatment should be further investigated through clinical studies aimed to establish a correct irrigation protocol. Within the limits of the study, the cryotreatment seems to have a greater effect on the reduction in bacteria compared to a standard NaOCl irrigation.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma (CTCL), has a poor prognosis and infections represent the most frequent cause of death. Polymorphonucleate granulocytes (PMNs) constitute an essential part of the innate immune system: their phagocytic and killing activity against pathogens is mediated by the interactions between Toll-like receptors (TLRs) and the Pathogen-associated molecular patterns (PAMPs). The aim of this study was to investigate PMN functional activity and phenotype in SS patients and their correlation with the onset of infectious complications. This prospective study enrolled 18 consecutive SS patients; PMN functional activity was evaluated by phagocytosis and intracellular killing tests towards Klebsiella pneumoniae. Flow-cytometry was applied to analyze PMN phenotype. PMNs from SS patients displayed a reduced phagocytic activity and intracellular killing against K. pneumoniae at 30 min and 60 min, more pronounced in SS patients with recurrent infections. CD11b and CD66b median fluorescence intensity (MFI) was significantly higher in SS than in healthy subjects, whereas CD62L MFI was decreased. No significant differences in TLR2, 4, 8 and 9 percentage expression or MFI were found. An increased TLR5 percentage expression was documented. The impairment in PMN functional activities in SS could favour the immune-suppression and raise infection risk.

Biomaterial-associated infection (BAI), a clinical problem resulting in septic failure of joint replacement implants, is initiated by bacterial adhesion, often by Staphylococcus epidermidis. Ultra high molecular weight polyethylene (UHMWPE) is a material of choice for joint replacement; reducing the adhesion of S. epidermidis to the polymer could be a means to decrease infection. We examined the adhesion of two ATCC and one clinical strain of S. epidermidis to standard polyethylene (PE), vitamin E blended UHMWPE (VE-PE), and oxidized UHMWPE (OX-PE) after different incubation times: a significant (p

In order to highlight the potential erythromycin immunomodulatory properties related to different antibiotic resistance patterns in Streptococcus spp., we evaluated the influence of the macrolide on the PMNs primary functions against erythromycin-susceptible (Ery-S) and erythromycin-resistant (Ery-R) S. pyogenes strains. A total of 438 S. pyogenes were isolated over the period 2005–2007. On the basis of the triple disk testing, 345 out of 438 S. pyogenes isolates were Ery-S and 93 were Ery-R; among the resistant strains, 65 displayed the cMLSB phenotype, 23 had the M phenotype and 5 had iMLSB phenotype. Concerning antibacterial activity of PMNs, our results showed that erythromycin did not modify bacterial uptake, but significantly increased the phagocyte intracellular killing, compared with controls, for both Ery-S and Ery-R strains. Consequently, this report underlines that in immunocompetent hosts the dichotomy between the in vitro resistance and clinical trial data for antimicrobial agents should be thoroughly re-evaluated.

We report a case of bronchopneumonia due to Schizophyllum commune in an immunocompromised patient. While this fungus rarely causes disease in humans, it has been reported in association with several clinical entities and lung disorders. A 59-year-old white man with a gastric carcinoma was admitted to S. Giovanni Battista Hospital (Turin, Italy). Three days after the admission, he developed a bronchopneumonia, which was diagnosed through the use of X-ray and showed an abnormal infiltrative shadow. Samples of bronchial aspirate were collected for laboratory microbiological investigation. Direct microscopic examination of these specimens revealed the presence of numerous septate, hyaline hyphae and rare clamp connections. Sabouraud Dextrose Agar and Columbia agar plus 5% blood media inoculated with portions of the same specimens yielded, after 4-5 days of incubation at 25 degrees C and 37 degrees C, a cottony white mould. The fungus was identified on the basis of its macroscopic and microscopic morphology. The macroscopic examination of the colony showed raised, curved, fan-shaped and shell-like basidiocarps. The microscope examination revealed the presence of hyaline, septate hyphae with clamp connections and short, thin spicules. The fungal isolate was identified as S. commune. The patient was cured after therapy with intravenous fluconazole (600 mg twice daily for over six weeks).

Dermatophytosis caused by Trichophyton rubrum is the most common cutaneous fungal infection in industrialized countries and worldwide with high recurrence and lack of treatment response. In addition, patients with cutaneous and concurrent toenail lesions are often misdiagnosed and therefore treated with an inappropriate therapy. In this study, we evaluated five previously misdiagnosed cases of T.rubrum chronic dermatophytosis sustained by two variants at sites distant from the primary lesion. Our patients were successfully treated by systemic and topical therapy, and 1 year after the end of therapy follow-up did not show any recurrence of infection.Our data indicate that the localization of all lesions, the isolation and the identification of the causative fungus are essential to establish the diagnosis and the setting of a correct therapeutic treatment to avoid recurrences.

Companion animals, often asymptomatic reservoir of fungi, can be important sources of infection in humans, due to the close contact with their owners. The present study was aimed to assess the occurrence of dermatophytes and other fungi isolated from pet dermatological lesions in Turin, Italy. Dermatological specimens were examined for fungal elements by direct microscopy and cultured to detect dermatophytes, other filamentous fungi and yeasts: 247 pets (118 cats, 111 dogs and 18 dwarf rabbits) were positive for fungal detection in culture. Microsporum canis was the most frequent dermatophyte in cats and dogs, whereas Trichophyton mentagrophytes was the most common in rabbits. Among the other fungi, for all examined pets, dematiaceous fungi were the most isolated, followed by Mucorales, penicilli, yeasts and yeast-like fungi, and aspergilli. No gender predisposition was detected for dermatophyte growth; on the contrary, for the other fungi male cats were more susceptible than female. The highest fungal occurrence was recorded in1-year-old cats for dermatophytes, and in5-year-old cats and dogs for the other fungi. Autumn was the period associated with a relevant incidence of fungal infection. Finally, fungi were more frequent in non pure-breed cats and in pure-breed dogs. These data underline the importance to timely inform pet owners about the potential health risk of infection caused not only by dermatophytes but also by non-dermatophyte fungi, routinely considered to be contaminants or harmless colonizers, since their role as source of zoonotic infections is not to be excluded.

ABSTRACT Background: Chronic wounds, characterized by hypoxia, inflammation and impaired tissue remodeling, are often worsened by bacterial/fungal infections. Intriguingly, chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLNs) have proven effective in delivering oxygen to hypoxic tissues. Aim: The present work aimed at investigating nanodroplet antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) or Candida albicans, toxicity on human keratinocytes (HaCaT) and ultrasound (US)-triggered transdermal delivery. Materials & methods: Nanodroplet antibacterial/antifungal properties, human cytotoxicity, and US-triggered transdermal delivery were measured through microbiological, biochemical, and sonophoresis assays, respectively. Results: OLNs and oxygen-free nanodroplets (OFNs) displayed short- or long-term cytostatic activity against MRSA or Candida albicans, respectively. OLNs were not toxic to keratinocytes, whereas OFNs slightly affected cell viability. Complementary US treatment promoted OLN transdermal delivery. Conclusion: As such, US-activated chitosan-shelled OLNs appear as promising, nonconventional and innovative tools for adjuvant treatment of infected chronic wounds.

The difficulties involved in detecting and enumerating Mycobacterium avium subsp. paratuberculosis (MAP) as a pathogen potentially involved in Crohn's disease (CD) are well known. This study aimed to improve this situation through the application of multiple laboratory diagnostic tests to detect and isolate this bacterium from different specimens collected from CD-patients and non-CD subjects as controls. A total of 120 samples (terminal ileum and colon biopsies, blood and stool) were obtained from 19 CD-patients and from 11 individuals who did not have a clinicopathological diagnosis of CD (non-CD controls) attending for ileocolonoscopy. All samples were processed by staining techniques, culture on both solid and liquid media, and Insertion Sequence 900/F57 real-time PCR. The MAP frequency in CD-patients was found in a significantly greater proportion than in non-CD subjects; the most positive samples were biopsies from CD-patients tested by real-time PCR. MAP detection in biopsies, and in the other samples, by applying multiple and validated laboratory diagnostic tests, could be a marker of active infection, supporting MAP involvement in CD.

Microorganism adhesion on polyethylene for total joint arthroplasty is a concern. Many studies have focused on vitamin E-stabilized ultrahigh-molecular-weight polyethylene (UHMWPE), whereas first-generation, highly crosslinked UHMWPE, which is the most commonly used in clinical practice, has been scarcely evaluated.We aimed (1) to compare the adherence of Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Candida albicans with virgin (untreated) UHMWPE (PE) and crosslinked UHMWPE (XLPE); (2) to correlate the results with the biomaterial surface properties; and (3) to determine whether the decreased adhesion on vitamin E-stabilized UHMWPE (VE-PE) previously recorded for bacteria can also be confirmed for C albicans.Microbial adhesion of biofilm-producing American Type Culture Collection (ATCC) and clinical strains on XLPE and VE-PE were compared with PE at 3, 7, 24, and 48 hours of incubation and quantified, as colony forming units (CFU)/mL, using a sonication protocol. Sample surfaces were characterized by scanning electron microscopy, roughness and contact angle measurements, attenuated total reflection-Fourier transform infrared spectroscopy, and x-ray photoelectron spectroscopy (XPS) to reveal qualitative differences in surface composition and topography that could influence the microbial adhesion. The results were analyzed by descriptive statistics and tested by unpaired t-tests.All microorganisms, both ATCC and clinical strains, showed lower adhesion (p0.05) on XLPE with adhesion percentages ranging from 18% to 25%, compared with PE with adhesion percentages ranging from 51% to 55%, after 48 hours. Only the ATCC S epidermidis showed a reduced adhesion profile even at 3 hours (adhesion ratio of 14% on XLPE versus 50% on PE) and 24 hours (19% on XLPE versus 55% on PE) of incubation. ATCC and clinical C albicans were less adherent to XLPE than to PE (p0.05) showing even at the earlier incubation time points adhesion values always of 10(3) CFU/mL and 10(4) CFU/mL, respectively. Roughness and contact angle were 0.8 ± 0.2 μm and 92° ± 3°, respectively, with no differences among samples. Qualitative differences in the surface chemical composition were revealed by XPS only. A confirmation of the decreased adhesion on VE-PE respect to PE was also registered here for C albicans strains (p0.05).Vitamin E stabilization and crosslinking of UHMWPE are capable of reducing microbial adhesion. Further studies are needed to fully elucidate the mechanisms of modulation of microbial adhesion to medical-grade UHMWPE.Our results suggest that VE-PE and XLPE may have an added benefit of being more resistant to bacterial adhesion, even fungal strains.

It is known that antimicrobial agents possess several, beneficial, secondary properties which complement their primary antimicrobial activity like the immunomodulatory capacity that enforces host defense mechanisms or reduces host inflammatory response. In this review the current state of our recent research about the interaction between some antimicrobial agents and the immune system as complex pyramid of redundant cellular factors, humoral effectors and mediators against various microbial pathogens, will be presented and compared with recent literature data. The nature of such interactions is diverse and depends on the drug, the host immunological status and the microorganism. A more complete understanding of the host immune modulation by antimicrobial drugs may guide the selection of appropriate regimens for given clinical situations.

Objective In this study the potential presence of bacteria in radicular cyst (RC) and keratocystic odontogenic tumour(KCOT) fluids from clinically asymptomatic patients was investigated. Materials and methods Cyst fluids were collected by needle aspiration from 16 patients with asymptomatic osteolytic lesions (10 RCs and 6 KCOTs) undergoing surgery. All samples were transferred into tubes containing pre-reduced transport medium, delivered to the microbiology laboratory and processed within 1 h. The cysts, surgically enucleated, were sent for standard histopathological examination. Cyst fluid samples were cultured on selective and differential media in anaerobic (for about 2 weeks) and aerobic (for 24–48 h) conditions to detect viable microorganisms. After incubation, the colonies were counted, Gram-stained and identified by biochemical tests. Results Cultures were positive for the presence of bacteria in 15 (9 RCs, 6 KCOTs) out of 16 cases. RCs and KCOTs generally yielded low bacterial counts (102–104 CFU/ml) and were predominantly colonized by obligate anaerobes (64%), whereas less commonly by facultative anaerobes (36%). No significant differences in the detection frequencies of obligate and facultative anaerobes were evidenced between RCs and KCOTs. Propionibacterium acnes was the most common obligate anaerobe recovered both in RC and KCOT fluids. Among facultative anaerobes, Gemella morbillorum was more frequently isolated in KCOTs, whereas Staphylococcus spp. in RCs. Conclusions Bacteria may be present and persist within fluids of clinically asymptomatic jaw cystic lesions. The influence of bacteria and latent bacterial infection within cystic jaw lesions should be reconsidered in odontogenic cyst progression.

Background: Atopic dermatitis (AD) patients present an high susceptibility to infections. The phagocytic activity of polymorphonuclear granulocytes (PMNs) is mediated by the interactions between Toll-like receptors (TLRs) and pathogen-associated molecular patterns. Objective: To investigate functional activity and phenotype of PMNs in AD patients. Methods: In vitro PMN phagocytosis and intracellular killing towards Klebsiella pneumoniae were evaluated in 24 AD patients; flow cytometry was applied to analyze PMN phenotype. Results: PMNs from AD patients displayed both reduced phagocytic activity and intracellular killing against K. pneumoniae than healthy subjects (HS). CD11b, CD66b, TLR2, TLR4 and TLR5 median fluorescence intensity (MFI) on PMN membrane were significantly higher in AD patients than in HS. Conclusion: PMN functional impairment in AD patients could represent an additional cause of skin infections, coupled with other known defects in the innate immune system. The increased MFI of adhesion molecules and TLRs is rather a consequence of the increased skin barrier permeability to bacterial molecules capable of stimulating immunological reactions.

The essential oils have started to be recognized for their potential antimicrobial role only in recent years. Clinical experience showed that the efficacy of antimicrobial agents depends not only on their direct effect on a given microorganism but also on the functional activity of the host immune system. Since data on the effects of essential oils on the innate immune system are scanty and fragmentary, the aim of this study was to evaluate the influence of thyme (red) essential oil (EO), at subinhibitory/inhibitory concentrations, on intracellular killing activity by human polymorphonuclear granulocytes (PMNs) against Candida albicans. In order to provide a frame of reference for the activity of this EO, its in vitro killing activity in the absence of PMNs was also evaluated. Results showed that EO at subminimal inhibitory (subMIC)/minimal inhibitory (MIC) concentrations significantly enhanced intracellular killing of C. albicans in comparison with EO-free controls and was comparable to the positive control (fluconazole). In in vitro killing assays without PMNs, we observed progressive growth of the yeast cells in the presence of EO subMIC/MIC concentrations. A positive antifungal interaction with phagocytes could explain why this EO, which appeared to be only fungistatic in time-kill assays, had efficacy in killing yeast cells once incubated with PMNs.

Phagocyte-dependent cellular immunity in chronic kidney disease patients undergoing haemodialysis treatment is frequently impaired owing to the uraemic state, resulting in an intrinsic susceptibility to developing invasive fungal infections with high mortality rates. Since synergism between phagocytic cells and antifungal drugs may be crucial for successful therapy, the aim of this study was to evaluate the effects exerted by caspofungin (CAS) on the functional activities of polymorphonuclear cells (PMNs) in haemodialysed patients (HDs) towards Candida albicans compared with those of PMNs from healthy subjects (HSs). PMNs were separated from venous blood samples of 66 HDs and 30 HSs (as controls), and measurement of phagocytic and intracellular fungicidal activities of HD-PMNs and HS-PMNs was performed in the presence of CAS at the minimum inhibitory concentration (MIC) and at sub-MICs. CAS-free controls were also included. In the drug-free test condition, no significant difference between the phagocytic activity of HD-PMNs and HS-PMNs was detected. In contrast, a progressive decline in the intracellular killing activity of HD-PMNs against proliferating yeasts was observed. CAS at MIC and sub-MIC levels was able to improve significantly the intracellular fungicidal activity of HD-PMNs against C. albicans, restoring their functionality. These findings provide evidence that CAS exerts a synergistic effect on HD-PMNs against C. albicans, being able to strength the depressed intracellular killing activity. These results corroborate the use of CAS as an effective therapeutic option for the treatment of invasive fungal infections in HDs, in whom even a marginal influence of antifungal drugs on host response may have a relevant effect.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease and may contribute to the onset and development of Crohn’s disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.

This study aimed to compare the caspofungin immunomodulating activities against Candida albicans on polymorphonuclear cells (PMNs) from renal transplant recipients (RTRs) and healthy subjects (HSs). RTR PMNs showed a significantly reduced fungicidal activity compared with that of HS PMNs. Addition of caspofungin to RTR PMNs significantly potentiated the yeast intracellular killing rate, achieving values similar to those observed for HS PMNs. These data show that caspofungin is suitable for invasive candidiasis treatment in patients with immune system-impaired components.

The influence of caspofungin on polymorphonuclear leukocyte (PMN) phagocytosis and intracellular killing of Candida albicans was investigated. Caspofungin, at all of the concentrations tested (2, 3.2, and 8 μg/ml), significantly increased intracellular killing by PMNs through its direct action on both yeast cells and PMNs, indicating the potential ability of caspofungin to synergize with phagocytes for candidal killing. Caspofungin may therefore constitute an effective therapeutic option for the treatment of invasive fungal infections, including those refractory to conventional treatment with azole agents.

To evaluate the synergistic activity of erythromycin and human polymorphonuclear cells (PMNs) on the binomial erythromycin-resistant (ERY(R)) Streptococcus pyogenes/host, the phagocytic and bactericidal activities of PMNs against ERY(R) streptococcal strains (cMLS(B), M, and iMLS(B) A, B and C phenotypes) were assessed in the presence of the macrolide. The results showed that when erythromycin, PMNs and streptococci [both erythromycin-sensitive (ERY(S)) and ERY(R)] were simultaneously present in the culture medium, PMN phagocytic activity was similar to that of drug-free controls. In contrast, the results emphasised a significant high increase in intracellular killing by PMNs in the presence of erythromycin not only for ERY(S) streptococci but also for ERY(R)S. pyogenes with high (cMLS(B), iMLS(B) A and iMLS(B) B phenotypes) and moderate (M and iMLS(B) C phenotypes) erythromycin resistance compared with controls without drug. From literature data it emerged that, even if intracellularly concentrated, erythromycin is relatively inactive because of its instability. The results indicate that the enhanced intra-PMN streptococcal killing detected is mainly attributable to PMN bactericidal systems that synergise with intracellular erythromycin in eradicating ERY(R)S. pyogenes strains (both with high and moderate resistance). These data confirm that the antibiotic resistance detected in vitro does not always imply a failure of antimicrobial treatment.

The increased prevalence of dermatomycoses along with the wide range of organisms now recognized as potential pathogens needs accurate laboratory isolation and identification of the aetiological agents. In this report three cases of foot dermatomycoses due to filamentous fungi commonly present in the environment with ubiquitous distribution are described in immunocompetent subjects. Skin and nail samples were collected, suspended in 20% KOH solution, examined under a light microscope and cultured in Mycobiotic agar and Sabouraud dextrose agar containing chloramphenicol to detect fungal growth. Phoma herbarum, Chaetomium globosum, and Microascus cinereus were isolated and identified.

Antimicrobial agents and polymorphonuclear cells (PMNs) have the potential to interact in such a way that improve the therapy for infectious diseases. In immunocompromised patients highly susceptible to microbial infections with high morbidity and mortality, several metabolic and functional alterations in PMNs, mostly related to microbicidal activity, are observed. Therefore, the antibiotic of choice should have a good antimicrobial effect without impairing host defences. The aim of this study is to evaluate in vitro effects of sub-inhibiting fosfomycin tromethamine (FT) concentrations on the primary functions of PMNs from healthy subjects and immunocompromised patients (haemodialysed and renal transplant recipients), against an ESBL-producing Escherichia coli, the most common aetiological agent in urinary tract infections (UTIs). FT is considered a first line drug in the eradication of UTIs due to its appropriate antimicrobial spectrum, oral bioavailability and minimal risk of microbial resistance. Our results provide evidence that FT is able to induce enhancement of the depressed phagocytic response of PMNs from patients on chronic haemodialysis and from renal transplant recipients, restoring their primary functions in vitro against ESBL-producing E. coll All these data permit the conclusion that uremic-infected patients might additionally benefit from the immunomodulating properties of FT.

Aims: This study compared the in vitro activity of telithromycin with that of azithromycin against 438 Streptococcus pyogenes and 198 Streptococcus pneumoniae, isolated over the period 2005–2007 from specimens of different human origin obtained in three Piemonte Region’s hospitals. Methods and Results: The determination of antimicrobial activity was evaluated by the microdilution broth method and the erythromycin-resistant (Ery-R) phenotypes by the triple-disc test. Exactly 78·8% of S. pyogenes and 69·2% of S. pneumoniae were erythromycin-susceptible (Ery-S). Concerning S. pyogenes, telithromycin was active against M and inducible MLSB, subtype-C, phenotypes but not against constitutive MLSB strains. Telithromycin acted well against all S. pneumoniae, irrespective of their mechanism of macrolide-resistance. On the contrary, the Ery-R isolates, both S. pyogenes and S. pneumoniae, were resistant to azithromycin. Conclusions: Our results indicate that macrolide resistance in streptococci still persist in northwest Italy (21·2% of S. pyogenes and 30·8% of S. pneumoniae) and that telithromycin is confirmed as being extremely active even against recent clinical Ery-R streptococcal isolates. Significance and Impact of the Study: The present study emphasizes that an active surveillance of the phenotype distribution and antibacterial resistance in streptococci is essential in guiding the effective use of empirical treatment option for streptococcal infections, also at regional level.

Purpose Sutures used in oral surgery should avoid or limit bacterial adhesion and proliferation to those parts exposed to oral fluids. Hence, microbial colonization on various intraoral suture materials from patients undergoing dental surgery was compared. Patients and Methods During dentoalveolar surgery, various suture materials were used in 60 patients, who were randomly divided into 5 groups of 12. In each group, silk was placed intraorally in association with a different type of suture (ie, Supramid, Synthofil, Ethibond Excel, Ti-cron, Monocryl) at the same site to compare microbial colonization intraindividually. Eight days postoperatively, the sutures were removed, and adhered micro-organisms were isolated, counted, and identified through enzymatic activities and fermentation of sugars. Results In all 60 patients, silk sutures exhibited the smallest affinity toward the adhesion of bacteria compared with considerable proliferation with nonresorbable multifilament sutures (Supramid, Synthofil, Ethibond Excel, Ti-cron). On the contrary, the microbial load was significantly lower when absorbable monofilament Monocryl was used. A greater quantity of bacteria was found on nonresorbable sutures than on absorbable ones, and nearly 2 times more facultative anaerobic bacteria were isolated in total. Conclusions Our results show that bacteria adhere with different affinity to various types of suture materials. Absorbable silk and Monocryl exhibited the smallest number of adherent bacteria. Colonization by pathogens on sutures leads to the recommendation that sutures should be removed as early as possible after surgery is performed, to eliminate or to limit the reservoir for oral pathogens. This recommendation is dependent on whether the suture is absorbable.

This report documents tinea pedis and tinea unguium in a 7-year-old child. In all cultures Trichophyton rubrum was present. As tinea pedis and tinea unguium affect adults more often than children, they might be overlooked and misdiagnosed in the latter.

It has been previously demonstrated that some antimicrobial agents enhance activities of human polymorphonuclear neutrophils (PMNs). The effect on the release of cytokines in an inflammatory context from PMNs by these antibiotics was evaluated. We studied the effect of the release of some cytokines by human PMNs RT-PCR analysis on a clinical strain of Klebsiella pneumoniae by comparing the effect with that observed in the presence of co-amoxiclav, sanfetrinem, clarithromycin, prulifloxacin and tobramycin. All the drugs tested were capable of modulating PMN synthesis in vitro of pro-inflammatory cytokines IL-8, IL-1beta, TNF-alpha and IL-6, but not that of anti-inflammatory cytokine IL-10. The degree of their stimulatory or inhibitory potency varied with the cytokine examined.

Current antibiotic therapy encourages the use of antibiotics that may potentiate the host's immune defences. We therefore investigated the effect of thiamphenicol (TAP), the active principle of thiamphenicol glycinate acetylcysteinate (TGA), on human granulocyte functions, mainly phagocytosis and intracellular killing of Streptococcus pyogenes. Our findings support the use of thiamphenicol in the treatment of respiratory tract infections caused by S. pyogenes as it acts directly against the pathogen as well as in cooperation with PMNs by eliciting their intracellular killing.

Allergic rhinitis is known to be one of the most common chronic diseases in the industrialized world. According to the concept that allergic rhinitis patients generally suffer from an immune deficit, in order to stimulate specifically or aspecifically their immune system, immunomodulating agents from various sources, such as synthetic compounds, tissue extracts or a mixture of bacterial extracts, have been used. The aim of the present trial is to evaluate the efficacy of the treatment with an immunostimulating vaccine consisting of a polyvalent mechanical bacterial lysate (PMBL) in the prophylaxis of allergic rhinitis and subsequently to analyze its in vivo effects on immune responses. 41 allergic rhinitis patients were enrolled: 26 patients were randomly assigned to the group for PMBL sublingual treatment and 15 others to the group for placebo treatment. For all 26 patients blood samples were drawn just before (T0) and after 3 months of PMBL treatment (T3) to evaluate plasma IgE levels (total and allergen-specific) and the cytokine production involved in the allergic response (IL-4, IFN-γ). The results of our study indicate that PMBL is effective in vivo in the reduction or in the elimination of the symptoms in rhinitis subjects during the treatment period in comparison to a non-immunostimulating treatment. A significant and clinically relevant improvement was found in 61.5%, a stationary clinical response was registered in 38.4% and no negative side effects associated with the medication or worsening were recorded. At the end of a 3-month follow up period the clinical picture remained the same as that observed at T3. PMBL treatment did not affect the serum IgE levels (either total or allergen-specific) and did not induce significant changes in IFN-γ concentration. In contrast, PMBL therapy may be accompanied, in some patients, by a potential immunomodulating activity by decreasing IL-4 cytokine expression.