Antimicrobial peptide human ß-defensin-2 improves in vitro cellular viability and reduces pro-inflammatory effects induced by enteroinvasive Escherichia coli in Caco-2 cells by inhibiting invasion and virulence factors' expression.

Escherichia coli is one of the commensal species most represented in the intestinal microbiota. However, there are some strains that can acquire new virulence factors that enable them to adapt to new intestinal niches. These include enteroinvasive E. coli (EIEC) that is responsible for the bacillary dysentery that causes severe diarrheal symptoms in both children and adults. Due to the increasing onset of antibiotic resistance phenomena, scientific research is focused on the study of other therapeutic approaches for the treatment of bacterial infections. A promising alternative could be represented by antimicrobial peptides (AMPs), that have received widespread attention due to their broad antimicrobial spectrum and low incidence of bacterial resistance. AMPs modulate the immune defenses of the host and regulate the composition of microbiota and the renewal of the intestinal epithelium. With the aim to investigate an alternative therapeutic approach, especially in the case of antibiotic resistance, in this work we created a line of intestinal epithelial cells able to express high concentrations of AMP human ß-defensin-2 (HBD-2) in order to test its ability to interfere with the pathogenicity mechanisms of EIEC. The results showed that HBD-2 is able to significantly reduce the expression of the proinflammatory cytokines by intestinal epithelial cells, the invasiveness ability of EIEC and the expression of invasion-associated genes.

Prevention of recurrent respiratory infections : Inter-society Consensus.

Recurrent respiratory infections (RRIs) are a common clinical condition in children, in fact about 25% of children under 1 year and 6% of children during the first 6 years of life have RRIs. In most cases, infections occur with mild clinical manifestations and the frequency of episodes tends to decrease over time with a complete resolution by 12 years of age. However, RRIs significantly reduce child and family quality of life and lead to significant medical and social costs.Despite the importance of this condition, there is currently no agreed definition of the term RRIs in the literature, especially concerning the frequency and type of infectious episodes to be considered. The aim of this consensus document is to propose an updated definition and provide recommendations with the intent of guiding the physician in the complex process of diagnosis, management and prevention of RRIs.

Total and Differential Cell Counts as a Tool to Identify Intramammary Infections in Cows after Calving.

Milk differential somatic cells count (DSCC), made possible under field conditions by the recent availability of a high-throughput milk analyzer may represent an improvement in mastitis diagnosis. While an increasing number of studies reports data on DSCC on individual cow samples, very few concerns DSCC from quarter milk samples. This paper reports for the first time the results of a retrospective study aiming to assess the performance of total (SCC), DSCC, and a novel calculated marker (PLCC) measured on quarter milk samples as a method to identify cows at risk for intramammary infection (IMI) in the first 30 days after calving. Overall, 14,586 valid quarter milk samples (3658 cows) taken in the first 30 days of lactation were considered. Quarters with major pathogens (MP) IMI, as expected, showed significantly higher means for SCC, DSCC, and PLCC. The accuracy, sensitivity, and specificity of the diagnosis based on different cut-offs calculated by ROC analysis are relatively close among DSCC, PLCC, and SCC (up to cut-off of 200,000 cells/mL). However, decision-tree analysis which includes the costs of analysis, but also the costs of the actions taken after test results showed as PLCC has the lowest cost among the three markers, and PLCC and SCC are cost effective when MP prevalence is higher than 6-10%. This diagnostic approach is of high interest particularly when selective dry cow therapy is applied to improve animal health at the herd level.

Updated Guidelines for the Management of Acute Otitis Media in Children by the Italian Society of Pediatrics: Treatment.

BACKGROUND: New insights into the diagnosis, treatment and prevention of acute otitis media (AOM) have been gained in recent years. For this reason, the Italian Paediatric Society has updated its 2010 guidelines. METHODS: A literature search was carried out on PubMed. Only pediatric studies published between January 1, 2010 and December 31, 2018 in English or Italian were included. Each included study was assessed according to the GRADE methodology. The quality of the systematic reviews was assessed using AMSTAR 2. The recommendations were formulated by a multidisciplinary panel of experts. RESULTS: Prompt antibiotic treatment is recommended for children with otorrhea, intracranial complications and/or a history of recurrence and for children under the age of 6 months. For children 6 months to 2 years of age, prompt antibiotic treatment is recommended for all forms of unilateral and bilateral AOM, whether mild or severe. Prompt antibiotic treatment is also recommended for children over 2 years with severe bilateral AOM. A watchful-waiting approach can be applied to children over 2 years with mild or severe unilateral AOM or mild bilateral AOM. High doses of amoxicillin, or amoxicillin-clavulanic acid for patients with a high risk of infection by Beta-lactamase producing strains, remain the first-line antibiotics. CONCLUSIONS: AOM should be managed on a case-by-case basis that takes account of the child's age, the severity of the episode and whether it is unilateral or bilateral. In patients under 2 years, prompt antibiotic treatment is always recommended.

The World Association against Infection in Orthopaedics and Trauma (WAIOT) procedures for Microbiological Sampling and Processing for Periprosthetic Joint Infections (PJIs) and other Implant-Related Infections.

While implant-related infections continue to play a relevant role in failure of implantable biomaterials in orthopaedic and trauma there is a lack of standardised microbiological procedures to identify the pathogen(s). The microbiological diagnosis of implant-related infections is challenging due to the following factors: the presence of bacterial biofilm(s), often associated with slow-growing microorganisms, low bacterial loads, previous antibiotic treatments and, possible intra-operative contamination. Therefore, diagnosis requires a specific set of procedures. Based on the Guidelines of the Italian Association of the Clinical Microbiologists (AMCLI), the World Association against Infection in Orthopaedics and Trauma has drafted the present document. This document includes guidance on the basic principles for sampling and processing for implant-related infections based on the most relevant literature. These procedures outline the main microbiological approaches, including sampling and processing methodologies for diagnostic assessment and confirmation of implant-related infections. Biofilm dislodgement techniques, incubation time and the role of molecular approaches are addressed in specific sections. The aim of this paper is to ensure a standardised approach to the main microbiological methods for implant-related infections, as well as to promote multidisciplinary collaboration between clinicians and microbiologists.

Bacterial contamination of saline nasal irrigations in children: An original research.

Microbiologic analysis of nasal saline irrigations (NSIs) used in hospitalized children was performed. Of 253 collected samples, 24.9% were positive, and the number of positive samples significantly increased over time (P < .001). Staphylococcus aureus was the most frequently detected bacterium (28.6%). None of the 118 patients who received NSIs developed a nasosinusal infection. Colonization by cutaneous and environmental germs is frequent and develops early. Hygienic measures should be advocated to reduce contamination.

Antibiotic sensitivities of coagulase-negative staphylococci and Staphylococcus aureus in hip and knee periprosthetic joint infections: does this differ if patients meet the International Consensus Meeting Criteria?

INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the main pathogens responsible for prosthetic joint infections (PJIs). As normal inhabitants of human skin, it is often difficult to define if they are contaminants, or if they have an active role in initiating infection. This study aims to evaluate differences in CoNS organisms (Staphylococcus hominis, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus warneri) and Staphylococcus aureus in terms of isolation rate and antimicrobial susceptibility from patients who met the International Consensus Meeting (ICM) criteria for PJIs and those who did not. METHODS: Staphylococci isolates from January 2014 to December 2015 retrieved from patients undergoing revision joint arthroplasty were classified in accordance with criteria established by the ICM of Philadelphia. RESULTS: As per the consensus classification, 50 CoNS and 39 S. aureus infections were recognized as pathogens, while 16 CoNS and four S. aureus were considered as contaminants. Frequency of isolation of S. aureus was significantly higher in infected patients than in those without infection, while no significant differences were observed among CoNS. Resistance to levofloxacin, erythromycin, gentamicin trimethoprim/sulfamethoxazole, and rifampicin was significantly more frequent in S. haemolyticus than in the other species, as well as resistance to erythromycin and gentamicin in S. hominis. In comparison to S. aureus, CoNS were significantly more resistant to daptomycin and gentamicin and more susceptible to rifampicin. CONCLUSION: CoNS, other than Staphylococcus epidermidis, are frequently isolated from PJIs, and their infective role and antimicrobial susceptibility need to be assessed on an individual patient basis. S. haemolyticus seems to emerge as responsible for PJI in a large volume of patients, and its role needs to be further investigated, also considering its pattern of resistance.

BAG-S53P4 as bone graft extender and antimicrobial activity against gentamicin- and vancomycin-resistant bacteria.

AIM: To evaluate the suitability of bioactive glass (BAG)-S53P4 as a bone-graft extender for large bony defect filling in bone and joint infection. MATERIALS & METHODS: Antimicrobial activity of BAG-S53P4 against clinically relevant strains isolated from bone and joint infections was evaluated by means of time-kill curves in presence of bone graft. Furthermore, the susceptibility to BAG of strains resistant to vancomycin and gentamicin was assessed. RESULTS: Though attenuated, BAG maintains a good in vitro antimicrobial activity in presence of human body fluids and tissues contained in bone graft, with the exception of Enterococcus faecalis. CONCLUSION: BAG-S53P4 is a suitable bone substitute that can be used as an extender with autologous bone graft to promote better fusion and healing.

In vitro comparison between α-tocopheryl acetate and α-tocopheryl phosphate against bacteria responsible of prosthetic and joint infections.

Biofilm-related infections represent a recurrent problem in the orthopaedic setting. In recent years, great interest was directed towards the identification of novel molecules capable to interfere with pathogens adhesion and biofilm formation on implant surfaces. In this study, two stable forms of α-tocopherol, the hydrophobic acetate ester and the water-soluble phosphate ester, were tested in vitro as coating for titanium prosthesis. Antimicrobial activity against microorganisms responsible of prosthetic and joints infections was assessed by broth microdilution method. In addition, α-tocopherol esters were evaluated for both their ability to hamper bacterial adhesion to and biofilm formation on sandblasted titanium surfaces. Results showed that only α-tocopheryl phosphate displayed antimicrobial activity against the tested strains. Both esters were able to significantly interfere with bacterial adhesion and to prevent biofilm formation, especially by Staphylococcus aureus and Staphylococcus epidermidis. The activity of α-tocopheryl phosphate was greater than that of α-tocopheryl acetate. Alterations at membrane levels have been reported in literature and may be likely responsible for the interference on bacterial adhesion and biofilm formation shown by α-tocopherol esters. Although further studies are needed to better investigate the mechanisms of action and the spectrum of activity of α-tocopherol esters, these characteristics together with the positive effect on wound healing and immune response, make these molecules promising candidate for coating in order to prevent implant-associated infections.

Erythritol/chlorhexidine combination reduces microbial biofilm and prevents its formation on titanium surfaces in vitro.

BACKGROUND: The purpose of this in vitro study was to evaluate the antibiofilm activity of a novel air-polishing powder consisting of erythritol and chlorhexidine, assessing its ability to reduce previously grown microbial biofilm and to prevent biofilm formation on titanium surfaces. METHODS: Clinical strains of Staphylococcus aureus, Pseudomonas aeruginosa, Bacteroides fragilis and Candida albicans isolated from peri-implantitis lesions were used. Biofilm was grown on sandblasted titanium discs and treated with erythritol/chlorhexidine. The antimicrobial activity was evaluated by determining the minimum inhibitory concentration and the minimum microbicidal concentration. The antibiofilm activity was assessed by semiquantitative spectrophotometric assay and by confocal laser scanning microscopy. RESULTS: Erythritol/chlorhexidine displayed an inhibitory and a microbicidal activity against all the tested strains. The spectrophotometric analysis showed that the treatment was effective in both reducing the previously developed biofilm and decreasing biofilm formation on titanium surfaces. Confocal laser scanning microscopy analysis showed a significant reduction of the total biofilm volume, with an increase of the percentage of dead cells of all the microorganisms tested. CONCLUSIONS: Erythritol/chlorhexidine displayed significant antimicrobial and antibiofilm activity against microorganisms isolated from peri-implantitis lesions. Due to its properties, it might represent a promising approach for the prevention and treatment of peri-implant diseases associated to microbial biofilm infections.

Ability of Lactobacillus kefiri LKF01 (DSM32079) to colonize the intestinal environment and modify the gut microbiota composition of healthy individuals.

BACKGROUND: Probiotics have been observed to positively influence the host's health, but to date few data about the ability of probiotics to modify the gut microbiota composition exist. AIMS: To evaluate the ability of Lactobacillus kefiri LKF01 DSM32079 (LKEF) to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. METHODS: Twenty Italian healthy volunteers were randomized in pre-prandial and post-prandial groups. Changes in the gut microbiota composition were detected by using a Next Generation Sequencing technology (Ion Torrent Personal Genome Machine). RESULTS: L. kefiri was recovered in the feces of all volunteers after one month of probiotic administration, while it was detected only in three subjects belonging to the pre-prandial group and in two subjects belonging to the post-prandial group one month after the end of probiotic consumption. After one month of probiotic oral intake we observed a reduction of Bilophila, Butyricicomonas, Flavonifractor, Oscillibacter and Prevotella. Interestingly, after the end of probiotic administration Bacteroides, Barnesiella, Butyricicomonas, Clostridium, Haemophilus, Oscillibacter, Salmonella, Streptococcus, Subdoligranolum, and Veillonella were significantly reduced if compared to baseline samples. CONCLUSION: L. kefiri LKF01 showed a strong ability to modulate the gut microbiota composition, leading to a significant reduction of several bacterial genera directly involved in the onset of pro-inflammatory response and gastrointestinal diseases.

Antibiofilm activity of sandblasted and laser-modified titanium against microorganisms isolated from peri-implantitis lesions.

Infections due to biofilm-producing microorganisms are one of the main causes for the failure of dental implants. Increasing efforts have been made in order to develop new strategies to prevent biofilm formation. In this study, the biofilm development on a newly designed laser-modified titanium implant surface was evaluated and compared to that on conventional sandblasted titanium used in implant dentistry. The amount of biofilm produced by Staphylococcus aureus, Pseudomonas aeruginosa and Porphyromonas gingivalis isolated from peri-implantitis was assessed by a semi-quantitative spectrophotometric method and by confocal laser scanning microscopy. Results showed a lower biofilm production on laser-modified surface compared to the sandblasted one. In particular, a significantly lower total volume of the biomass was observed on laser-modified surface, while no significant changes in live/dead bacteria percentages were noticed between materials. Modifying the topography of the conventional implant surface with laser ablation could represent a promising approach for inhibiting biofilm formation.

Antibiofilm agents against MDR bacterial strains: is bioactive glass BAG-S53P4 also effective?

OBJECTIVES: The treatment of bone and joint infections is challenging due to the presence of bacterial biofilm and the increasing emergence of multiresistant strains. BAG-S53P4 is a bone substitute that is characterized by osteoconductive and antimicrobial properties. The aim of this study was to assess the effectiveness of BAG-S53P4 against biofilm produced in vitro by multiresistant bacterial strains. METHODS: Multiresistant Staphylococcus epidermidis, Acinetobacter baumannii and Klebsiella pneumoniae isolated from bone and joint infections were used in this study. Titanium discs covered by bacterial biofilm were incubated with BAG-S53P4 or inert glass as a control. The amount of biofilm on each titanium disc was evaluated after 48 h of incubation by means of confocal laser scanning microscopy. RESULTS: Significantly lower total biomass volumes were observed for all strains after treatment with BAG-S53P4 when compared with controls. Moreover, the percentage of dead cells was significantly higher in treated samples than in controls for all the tested strains. CONCLUSIONS: BAG-S53P4 is able to reduce the biofilm produced by multiresistant S. epidermidis, A. baumannii and K. pneumoniae on titanium substrates in vitro, probably by interfering with cell viability. Owing to its osteoconductive, antibacterial and antibiofilm properties, the use of BAG-S53P4 may be a successful strategy for the treatment of bone and prosthetic joint infections.

Antimicrobial activity and resistance selection of different bioglass S53P4 formulations against multidrug resistant strains.

AIMS: This study aimed to evaluate the antimicrobial activity of two different formulations of bioglass BAG-S53P4 against multiresistant microorganisms involved in bone infections, and the capability of bioglass to select for resistance. METHODS: Antibacterial activity was evaluated by means of killing curves. The ability to select for resistant bacteria was evaluated by subculturing microorganisms in serial dilutions of bioglass. Scanning electron microscope acquisitions were conducted to evaluate bioglass-induced morphology changes. RESULTS: BAG-S53P4 formulations display a high antimicrobial activity and do not seem to select for resistance. Scanning electron microscopy analysis showed cell shrinkage and membrane damage after exposure to bioglass. CONCLUSIONS: BAG-S53P4 has a significant potential as bone substitute for the treatment of infections caused by multiresistant microorganisms.

May osteoarticular infections be influenced by vitamin D status? An observational study on selected patients.

BACKGROUND: Vitamin D deficiency has been associated with a high number of health outcomes, and its role on the immune system has been deeply investigated in recent years, although poor data are still available on vitamin D status in orthopedic infections including those of prosthetic implants. METHODS: We focused on preoperative values of 25(OH)D in selected groups of patients with septic (Group A) or aseptic (Group B) prosthetic loosening, infective bone disease such as septic arthritis and osteomyelitis (Group C) and other orthopedic pathologies (Group D) to evaluate differences in the vitamin D status. RESULTS: A high prevalence of vitamin D deficiency was recorded among the study population (16.5 ± 5.4 ng/mL, mean ± SD). Interestingly, all patients with an infection presented a higher 25(OH)D concentration (17.7 ± 5.3 ng/mL) in respect to uninfected ones (15.1 ± 5.6 ng/mL). Significantly higher levels of 25(OH)D were observed in patients with prosthetic joint infection (18.5 ± 6.5 ng/mL), when compared with those presenting an aseptic loosening (13.6 ± 9.4 ng/mL). CONCLUSIONS: Deficiency in vitamin D levels have been found in orthopaedic patients. Prosthetic joint infections seems to be associated to higher values of vitamin D in respect to other bone infections or to other orthopaedic conditions requiring surgery. More studies are needed to improve the knowledge on vitamin D status in these patients and to better clarify the role of vitamin D in relation to osteoarticular infections.

Nanotechnology approaches for antibacterial drug delivery: Preparation and microbiological evaluation of fusogenic liposomes carrying fusidic acid.

Many antibacterial drugs have some difficulty passing through the bacterial cell membrane, especially if they have a high molecular weight or large spatial structure. Consequently, intrinsic resistance is shown by some bacterial strains. Reduced cell membrane permeability is one of the mechanisms of resistance known for fusidic acid (FUS), a bacteriostatic steroidal compound with activity limited to Gram-positive bacteria. Moreover, the lipophilic character of FUS has been shown to cause drug retention inside the bilayers of cell membranes, preventing its diffusion towards target sites inside the cytoplasm. Targeting antimicrobial agents by means of liposomes may be a valid strategy in the treatment of infections refractory to conventional routes of antimicrobial treatment. On this basis, loading of FUS in fusogenic liposomes (FLs) was planned in this study. Fusogenic small unilamellar vesicles loaded with FUS were produced to evaluate their influence on improving the cell penetration and antibacterial activity of the antibiotic. The produced carriers were technologically characterised and were subjected to an in vitro microbiological assay against several strains of Gram-negative and Gram-positive bacteria. The experimental results showed that encapsulating FUS in a liposomal carrier can improve antimicrobial efficacy and reduce the effective concentration required, probably through putative mechanisms of increased diffusion through the bacterial cell membrane. In fact, whilst free FUS was active only on the tested Gram-positive strains, incubation of FUS-loaded FLs exhibited growth inhibitory activity both against Gram-positive and Gram-negative strains. The lowest MICs were obtained against Staphylococcus epidermidis (≤0.15 µg/mL) and Acinetobacter baumannii (37.5 µg/mL) clinical strains.

Colonization by methicillin resistant staphylococci of nares and skin in healthcare workers: a pilot study in spinal surgeries.

INTRODUCTION: Staphylococcus aureus and coagulase-negative staphylococci (CoNS) colonization among healthcare workers (HCWs) may have implications in development of infections and in spreading of resistance. This study aimed to determine the rate of methicillin-resistant staphylococci carriage in HCWs of spinal surgeries in an Italian Orthopaedic Institute. MATERIALS AND METHODS: Samples from nares, axillae and hands were inoculated onto appropriate media in order to perform colony counts of methicillin-susceptible and resistant S. aureus and CoNS. RESULTS: Prevalence of S. aureus and CNS was 42.3% and 98%, respectively. Methicillin-resistance was rather infrequent in S. aureus (13.5%) while it was detected in most of CoNS (90.4%). Methicillin resistant S. aureus were prevalently isolated from nares while axillae showed the highest methicillin-resistant CoNS colonization rates. CONCLUSIONS: A relatively high rate of methicillin resistant staphylococci was found among HCWs in spinal surgeries wards, thus evidencing the need for careful prevention measures and for periodic evaluation of spread among HCWs.

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.

Antiadhesive and antibiofilm activity of hyaluronic acid against bacteria responsible for respiratory tract infections.

To address the problem of limited efficacy of existing antibiotics in the treatment of bacterial biofilm, it is necessary to find alternative remedies. One candidate could be hyaluronic acid; this study therefore aimed to evaluate the in vitro antiadhesive and antibiofilm activity of hyaluronic acid toward bacterial species commonly isolated from respiratory infections. Interference exerted on bacterial adhesion was evaluated by using Hep-2 cells, while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet. Our data suggest that hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration-dependent manner, being notably active when assessed as pure substance. Moreover, we found that Staphylococcus aureus biofilm was more sensitive to the action of hyaluronic acid than biofilm produced by Haemophilus influenzae and Moraxella catarrhalis. In conclusion, hyaluronic acid is characterized by notable antiadhesive properties, while it shows a moderate activity against bacterial biofilm. As bacterial adhesion to oral cells is the first step for colonization, these results further sustain the role of hyaluronic acid in prevention of respiratory infections.

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.