Evaluation of a Modified Roche Enzymatic Ammonia Method for Roche Cobas 6000 (c501 Module) Automated Platform: When 5 µL Improves Performance.

BACKGROUND: We evaluated a modified Roche NH3L method developed by our group that significantly reduced the error flag "> ABS (> Absorbance)" on the COBAS 6000 (c501 module) automated platform. METHODS: Our study was finalized to validate the NH3L open method on COBAS 6000 (c501 module) with imprecision and correlation tests. In addition, the NH3L open method was evaluated for determination of lower limit of blank (LoB), lower limit of detection (LoD), and accuracy. RESULTS: The imprecision test showed good results with CV for all samples tested < 3 and < 5 for within-run and between-run assays. Correlation tests of NH3L classic and NH3L open method showed good correlation with R square = 0.95. "> ABS" obtained with the NH3L open were only 2% compared to NH3L classic method. CONCLUSIONS: Our study shows that the NH3L open method is reproducible and stable, providing values which correlate with those obtained by the traditional method. The ability to reduce the alarm > ABS by more than 95% thanks to lower background absorbance values makes this method reliable, avoiding re-testing or the need for sample dilutions.

Odontogenic and rhinogenic chronic sinusitis: a modern microbiological comparison.

BACKGROUND: Odontogenic sinusitis and sinonasal complications of dental disease or treatment (SCDDT) play a relevant, often underappreciated role in paranasal sinus infections. Treating SCDDT patients requires tailored medical and surgical approaches in order to achieve acceptable success rates. These approaches differ from common rhinogenic sinusitis treatment protocols mostly because of the different etiopathogenesis. Our study comprehensively evaluated microbiology and antibiotic resistance in SCDDT patients and compared findings with a control group of patients affected by rhinogenic sinusitis. METHODS: We performed microbiological sampling during surgery on 28 patients with SCDDT and 16 patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Colonies were isolated, Gram-stained, and the species identified using classic biochemical methods. These results were confirmed by DNA pyrosequencing, and then the resistance profile of each SCDDT isolate to various antibiotics was tested. RESULTS: Microbial growth was observed in all SCDDT patients, whereas samples from 60% of patients in the control group failed to yield any bacterial growth (p < 0.001). Anaerobes grew in 14% of SCDDT patients as compared to 7% of CRSwNP patients (p = 0.42). Of the isolates from SCDDT patients, 70% were susceptible to amoxicillin/clavulanate, whereas all isolates were susceptible to levofloxacin, teicoplanin, and vancomycin. Of the staphylococci identified, 80% were capable of producing beta-lactamase. CONCLUSION: Given the extent of microbiological contamination within the maxillary sinus of SCDDT patients, these infections should be regarded as a different class of conditions from rhinogenic sinusitis. Our findings support the need for different approaches in the treatment of SCDDT patients.

Soluble urokinase-type plasminogen activator receptor (suPAR) as new biomarker of the prosthetic joint infection: correlation with inflammatory cytokines.

Post-operative prosthetic joint infection (PJI) is the most common cause of failure of total joint arthroplasty, requiring revision surgery, but a gold standard for the diagnosis and the treatment of PIJ is still lacking. PJI is mainly due to Gram-positive bacteria, in particular, Staphylococcus Aureus, and more rarely by Gram-negative bacteria such as Pseudomonas. This study aimed to examine the diagnostic value of SuPAR in post-operative PJI, in order to explore the possible application of this new biomarker in the early diagnosis of PJI. The level of SuPAR has been measured in PJI patients and healthy controls, correlated with canonical inflammatory markers, such as C-reactive protein, IL-6, IL-1 and TNFα and the chemokine CCL2. Serum suPAR displayed a strongly significative increase in PJI patients compared to not infected controls, and a significative positive correlation with C-reactive protein, IL-6, IL-1 and TNFα and the chemokine CCL2. Also serum CCL2 showed statistically significative increase in PJI patients, and it displayed a strong positive correlation with serum suPAR. This study provides a clear indication of the diagnostic potential of suPAR, in association to routine inflammatory parameters such as CRP, in the diagnosis of PJI.

Treatment of atopic dermatitis eczema with a high concentration of Lactobacillus salivarius LS01 associated with an innovative gelling complex: a pilot study on adults.

GOALS: To evaluate the efficacy of a highly concentrated Lactobacillus salivarius preparation containing a gelling complex formed by Streptococcus thermophilus ST10 and tara gum in the treatment of atopic dermatitis (AD). BACKGROUND: Previous studies have demonstrated an improvement in AD symptoms after administration of the probiotic strain L. salivarius LS01. S. thermophilus ST10 and tara gum create a gelling complex that adheres to intestinal mucus and improves barrier function. STUDY: A prospective, controlled pilot trial was carried out to evaluate how the association of S. thermophilus ST10 and tara gum could improve the activity of L. salivarius LS01 administered at high doses to adults with AD. Twenty-five patients were included into the study: 13 were treated for 1 month with the active formulation, whereas 12 represented the placebo group. Scoring Atopic Dermatitis index was determined before and at the end of probiotic administration. Fecal samples were also collected to evaluate changes in bacterial counts of Staphylococcus aureus and clostridia. RESULTS: A significant improvement in SCORAD index was observed in the probiotic group after 1 month of treatment, whereas no significant changes occurred in placebo patients. A slight decrease in fecal S. aureus count was observed in probiotic-treated patients. CONCLUSIONS: Data obtained in this study suggest a potential role for L. salivarius LS01 in the treatment of AD. The addition of tara gum and S. thermophilus ST10 seems to improve the overall efficacy of the probiotic strain, in particular shortening the time required for the onset of the positive effects. Further studies to investigate the activity of this preparation are advisable.

Plasma components and platelet activation are essential for the antimicrobial properties of autologous platelet-rich plasma: an in vitro study.

Autologous platelet concentrates are successfully adopted in a variety of medical fields to stimulate bone and soft tissue regeneration. The rationale for their use consists in the delivery of a wide range of platelet-derived bioactive molecules that promotes wound healing. In addition, antimicrobial properties of platelet concentrates have been pointed out. In this study, the effect of the platelet concentration, of the activation step and of the presence of plasmatic components on the antimicrobial activity of pure platelet-rich plasma was investigated against gram positive bacteria isolated from oral cavity. The antibacterial activity, evaluated as the minimum inhibitory concentration, was determined through the microdilution two-fold serial method. Results seem to suggest that the antimicrobial activity of platelet-rich plasma against Enterococcus faecalis, Streptococcus agalactiae, Streptococcus oralis and Staphylococcus aureus is sustained by a co-operation between plasma components and platelet-derived factors and that the activation of coagulation is a fundamental step. The findings of this study may have practical implications in the modality of application of platelet concentrates.

Biofilm removal and antimicrobial activity of two different air-polishing powders: an in vitro study.

BACKGROUND: Biofilm removal plays a central role in the prevention of periodontal and peri-implant diseases associated with microbial infections. Plaque debridement may be accomplished by air polishing using abrasive powders. In this study, a new formulation consisting of erythritol and chlorhexidine is compared with the standard glycine powder used in air-polishing devices. Their in vitro antimicrobial and antibiofilm effects on Staphylococcus aureus, Bacteroides fragilis, and Candida albicans are investigated. METHODS: Biofilm was allowed to grow on sandblasted titanium disks and air polished with glycine or erythritol-chlorhexidine powders. A semiquantitative analysis of biofilm by spectrophotometric assay was performed. A qualitative analysis was also carried out by confocal laser scanning microscopy. Minimum inhibitory concentrations and minimum microbicidal concentrations were evaluated, together with the microbial recovery from the residual biofilm after air-polishing treatment. RESULTS: The combination of erythritol and chlorhexidine displayed stronger antimicrobial and antibiofilm activity than glycine against all microbial strains tested. CONCLUSION: Air polishing with erythritol-chlorhexidine seems to be a viable alternative to the traditional glycine treatment for biofilm removal.

Role and antimicrobial resistance of staphylococci involved in prosthetic joint infections.

PURPOSE: Staphylococci are responsible for approximately half of all prosthetic joint infections (PJIs) and they are often multi-drug resistant. The main purpose of this study was to evaluate the incidence of PJIs caused by staphylococci in our hospital from March 2010 to February 2012, with particular reference to antibiotic resistance in relation to their classification as contaminant or infecting isolates. METHODS: We analyzed samples recovered from 124 patients: most of them were male (55.8%) and the mean age was 66 ± 14 years. Prostheses derived from hip (54.8%) or knee (45.2%) replacement and they were processed by sonication. Isolates were identified using conventional biochemical methodologies. Antimicrobial susceptibility testing was carried out using the disk diffusion method as described by the European Committee on Antimicrobial Susceptibility Testing. RESULTS: A total of 135 staphylococci were isolated: the prevalent species was Staphylococcus aureus, but, on the whole, coagulase-negative staphylococci represented 57% of cases. Fifty-one isolates were recovered from a single sample and were therefore defined as contaminant. Linezolid and glycopeptides showed excellent activity versus all the tested isolates, while penicillin, levofloxacin, and erythromycin offered reduced activity against staphylococci. Interestingly, high resistance rates were observed for coagulase-negative staphylococci other than S. epidermidis classified as contaminant strains. CONCLUSIONS: We observed a remarkable spread of coagulase-negative staphylococci as causative agents of PJIs; but most of them were classified as contaminants. However, because of their low susceptibility to the antibiotics tested, further studies are necessary to evaluate their role as pathogens or as true contaminants.

In vitro antibiofilm activity of bioactive glass S53P4.

AIM: This work aimed to investigate the ability of different formulations of bioactive glass (BAG)-S53P4 to interfere with bacterial biofilm produced on prosthetic material by methicillin-resistant Staphylococcus aureus and multi-drug-resistant Pseudomonas aeruginosa. MATERIALS & METHODS: Antibiofilm activity of three formulations of bioglass was assessed at different time points through two different analyses: Crystal Violet and confocal laser scanning microscopy assays. RESULTS: Significant differences in the whole biofilm were observed between BAG-S53P4-treated and control samples, while no marked changes in antibiofilm activity were observed among the tested formulations. Data from colorimetric assay were confirmed by confocal laser scanning microscopy analysis, which evidenced the significant reduction in biomass and a decrease of total cell volume when both S. aureus and P. aeruginosa biofilms were treated with BAG-S53P4. CONCLUSION: BAG-S53P4 can be considered as an excellent adjuvant in the treatment of prosthetic infections related to biofilm.

Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material.

Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms.

Does PGE1 vasodilator prevent orthopaedic implant-related infection in diabetes? Preliminary results in a mouse model.

BACKGROUND: Implant-related infections are characterized by bacterial colonization and biofilm formation on the prosthesis. Diabetes represents one of the risk factors that increase the chances of prosthetic infections because of related severe peripheral vascular disease. Vasodilatation can be a therapeutic option to overcome diabetic vascular damages and increase the local blood supply. In this study, the effect of a PGE1 vasodilator on the incidence of surgical infections in diabetic mice was investigated. METHODOLOGY: A S. aureus implant-related infection was induced in femurs of diabetic mice, then differently treated with a third generation cephalosporin alone or associated with a PGE1 vasodilator. Variations in mouse body weight were evaluated as index of animal welfare. The femurs were harvested after 28 days and underwent both qualitative and quantitative analysis as micro-CT, histological and microbiological analyses. RESULTS: The analysis performed in this study demonstrated the increased host response to implant-related infection in diabetic mice treated with the combination of a PGE1 and antibiotic. In this group, restrained signs of infections were identified by micro-CT and histological analysis. On the other hand, the diabetic mice treated with the antibiotic alone showed a severe infection and inability to successfully respond to the standard antimicrobial treatment. CONCLUSIONS: The present study revealed interesting preliminary results in the use of a drug combination of antibiotic and vasodilator to prevent implant-related Staphylococcus aureus infections in a diabetic mouse model.

Toll-like receptor 2 in serum: a potential diagnostic marker of prosthetic joint infection?

Prosthetic joint infection (PJI) is a severe complication of arthroplasty and is still lacking diagnostic gold standards. PJI patients display high Toll-like receptor 2 (TLR2) serum levels, correlating with canonical inflammatory markers (C-reactive protein [CRP], interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α], and IL-1). Therefore, TLR2 serum levels could be considered a new potential diagnostic tool in the early detection of PJI.

Bioactive glass BAG-S53P4 for the adjunctive treatment of chronic osteomyelitis of the long bones: an in vitro and prospective clinical study.

BACKGROUND: This study aimed to explore the in vitro antibacterial activity of the bioglass BAG S53P4 against multi-resistant microorganisms commonly involved in osteomyelitis and to evaluate its use in surgical adjunctive treatment of osteomyelitis. METHODS: In vitro antibacterial activity of BAG-S53P4 against methicillin resistant Staphylococcus aureus and Staphylococcus epidermidis, Pseudomonas aeruginosa and Acinetobacter baumannii isolates was evaluated by means of time kill curves, with colony counts performed after 24, 48 and 72 hours of incubation. In vivo evaluation was performed by prospectively studying a cohort of 27 patients with a clinically and radiologically diagnosed osteomyelitis of the long bones in an observational study. Endpoints were the absence of infection recurrence/persistence at follow-up, no need for further surgery whenever during follow-up and absence of local or systemic side effects connected with the BAG use. RESULTS: In vitro tests regarding the antibacterial activity of BAG S53P4 showed a marked bactericidal activity after 24 hrs against all the tested species. This activity continued in the subsequent 24 hrs and no growth was observed for all strains after 72 hrs. Results of the clinical study evidenced no signs of infection in 24 patients (88.9%) at the follow-up, while 2 subjects showed infection recurrence at 6 months from index operation and one more needed further surgical procedures. BAG-S53P4 was generally well tolerated. CONCLUSIONS: The in vitro and in vivo findings reinforce previous observations on the efficacy of BAG-S53P4 for the treatment of chronic osteomyelitis of the long bones, also in the presence of multi-resistant strains and in immunocompromised hosts, without relevant side effects and without the need for locally adding antibiotics. TRIAL REGISTRATION: Deutschen Register Klinischer Studien (DRKS) unique identifier: DRKS00005332.

Use of dithiothreitol to improve the diagnosis of prosthetic joint infections.

Diagnosis of prosthetic joint infections (PJIs) remains a challenge for microbiologists, despite new techniques for bacteria isolation have been developed in recent years. A widely recognized standard method has not yet been indicated mainly because of limitations due difficult procedures and need of dedicated instrumentation. We evaluated the ability of a sulfhydryl compound routinely used in microbiology laboratories, dithiothreitol (DTT), to dislodge bacteria from biofilm, keeping them alive and cultivable for identification and antibiotic susceptibility testing. We compared DTT treatment against sonication of prosthesis and culture of periprosthetic tissues, in order to establish if it could be introduced in routine microbiological diagnosis of PJIs. The study was conducted on 76 patients, 34 with aseptic loosening of their prosthesis and 42 who were diagnosed for PJI. DTT treatment gave results similar to sonication in terms of bacterial yielding. Sonication provided higher sensitivity (71.4%) and specificity (94.1%) respect to periprosthetic tissue culture, while DTT showed the same specificity of sonication but a better sensitivity (85.7%), especially when the causative microorganism was Staphylococcus epidermidis. In conclusion, we demonstrated that DTT could be used for PJIs diagnosis, thanks to its ease of use and its high sensitivity and specificity.

A case of coinfection in a chronic maxillary sinusitis of odontogenic origin: identification of Dialister pneumosintes.

INTRODUCTION: In this report, we discuss the case of a 39-year-old woman presenting with a case of chronic maxillary sinusitis. METHODS: Dialister pneumosintes, Staphylococcus epidermidis, and Peptostreptococcus spp. were isolated from endosinusal samples obtained during surgery. The patient showed extensive periodontopathy and had undergone prior endodontic treatment for endodontic infection of teeth #13, #14, and #15, which failed and presumably acted as a bridge for the sinusal infection. After nasosinusal surgery, consisting of opening and toilet of the maxillary sinus, combined with extraction of the 3 previously mentioned teeth and antibiotic treatment, the patient showed complete healing. RESULTS: S. epidermidis and Peptostreptococcus spp. were identified with a traditional biochemical test and confirmed by pyrosequencing. Conversely, D. pneumosintes could not be identified with the conventional method, but it was identified using DNA pyrosequencing. In addition, to better understand the role and the virulence of this bacterium in odontogenic sinusitis, we have evaluated the ability of D. pneumosintes to produce biofilms onto inert surfaces. D. pneumosintes is a known endodontic and periodontal pathogen found in necrotic pulp, subgingival plaque, and deep periodontal pockets. CONCLUSIONS: To our knowledge, the pathogenic role of D. pneumosintes in odontogenic sinusitis has never been evidenced. Thus, its detection in endosinusal specimens may provide a significant insight into the pathogenesis of this relevant medical condition.

Antimicrobial activity of pure platelet-rich plasma against microorganisms isolated from oral cavity.

BACKGROUND: Autologous platelet concentrates (PCs) have been extensively used in a variety of medical fields to promote soft and hard tissue regeneration. The significance behind their use lies in the abundance of growth factors in platelets α-granules that promotes wound healing. In addition, antibacterial properties of PCs against various bacteria have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against oral cavity microorganisms such as Enterococcus faecalis, Candida albicans, Streptococcus agalactiae, Streptococcus oralis and Pseudomonas aeruginosa. Blood samples were obtained from 17 patients who underwent oral surgery procedures involving the use of P-PRP. The antibacterial activity of P-PRP, evaluated as the minimum inhibitory concentration (MIC), was determined through the microdilution twofold serial method. RESULTS: P-PRP inhibited the growth of Enterococcus faecalis, Candida albicans, Streptococcus agalactiae and Streptococcus oralis, but not of Pseudomonas aeruginosa strains. CONCLUSIONS: P-PRP is a potentially useful substance in the fight against postoperative infections. This might represent a valuable property in adjunct to the enhancement of tissue regeneration.

A case of a late and atypical knee prosthetic infection by no-biofilm producer Pasteurella multocida strain identified by pyrosequencing.

Prosthetic joint infections due to Pasteurella multocida are rarely but increasingly reported but no data on production of biofilm are available. We report the case of a woman with a late, haematogenous peri-prosthetic infection of cemented total knee arthroplasty caused by a strain of P. multocida identified by pyrosequencing and unable to produce biofilm. Comparison of clinical and laboratory findings with those reported in other patients evidenced differences mainly in the period of symptoms' onset and in the behaviour of some inflammatory markers.