Topical Treatment of Second-Degree Burn Wounds with Lactobacillus plantarum Supernatant: Phase I Trial.

Background & Objective: A burn wound is sterile immediately after injury, but opportunistic bacteria colonize the wound within 48 to 72 hours after the burn, causing delayed or failed burn wound healing. In addition, the presence of multidrug-resistant (MDR) pathogens doubles the treatment problems. Lactobacillus plantarum (L. plantarum) is a well-known antibacterial and healing agent that could be used topically to treat burn wounds. Case Series Presentation: This clinical trial study (Case Series) was performed on 20 patients with deep second-degree burns. Patients had bilateral wounds; the wound on one side of the body was considered as control (treated with silver sulfadiazine) and the other side of the body as treatment (treated with bacteria-free supernatants (BFS) of L. plantarum). The wounds were evaluated by microbial assessments and assessments related to healing. Pseudomonas aeruginosa, Klebsiella pneumonia, and Staphylococcus aureus were isolated from 4 (22.2%), 0%, and 2 (11.1%) of wounds treated with L. plantarum on the fifth day of the treatment, respectively. Furthermore, 12 (66.7%) of wounds treated with L. plantarum were free from bacteria. The need for skin grafting was the same in both treatment and control groups, but graft rejection in the group treated with L. plantarum was (0%) (P=0.02). Conclusion: Regarding eliminating or reducing infection and wound healing, bacteria-free supernatants of L. plantarum can be considered a possible topical treatment option in the case of second-degree burn wounds.

Biofilm formation and inflammatory potential of Staphylococcus saccharolyticus: A possible cause of orthopedic implant-associated infections.

Staphylococcus saccharolyticus, a coagulase-negative staphylococcal species, has some unusual characteristics for human-associated staphylococci, such as slow growth and its preference for anoxic culture conditions. This species is a relatively abundant member of the human skin microbiota, but its microbiological properties, as well as the pathogenic potential, have scarcely been investigated so far, despite being occasionally isolated from different types of infections including orthopedic implant-associated infections. Here, we investigated the growth and biofilm properties of clinical isolates of S. saccharolyticus and determined host cell responses. Growth assessments in anoxic and oxic conditions revealed strain-dependent outcomes, as some strains can also grow aerobically. All tested strains of S. saccharolyticus were able to form biofilm in a microtiter plate assay. Strain-dependent differences were determined by optical coherence tomography, revealing that medium supplementation with glucose and sodium chloride enhanced biofilm formation. Visualization of the biofilm by confocal laser scanning microscopy revealed the role of extracellular DNA in the biofilm structure. In addition to attached biofilms, S. saccharolyticus also formed bacterial aggregates at an early stage of growth. Transcriptome analysis of biofilm-grown versus planktonic cells revealed a set of upregulated genes in biofilm-embedded cells, including factors involved in adhesion, colonization, and competition such as epidermin, type I toxin-antitoxin system, and phenol-soluble modulins (beta and epsilon). To investigate consequences for the host after encountering S. saccharolyticus, cytokine profiling and host cell viability were assessed by infection experiments with differentiated THP-1 cells. The microorganism strongly triggered the secretion of the tested pro-inflammatory cyto- and chemokines IL-6, IL-8, and TNF-alpha, determined at 24 h post-infection. S. saccharolyticus was less cytotoxic than Staphylococcus aureus. Taken together, the results indicate that S. saccharolyticus has substantial pathogenic potential. Thus, it can be a potential cause of orthopedic implant-associated infections and other types of deep-seated infections.

Staphylococcus saccharolyticus Associated with Prosthetic Joint Infections: Clinical Features and Genomic Characteristics.

The anaerobic coagulase-negative staphylococcal species Staphylococcus saccharolyticus is a member of the normal skin microbiota. However, S. saccharolyticus is rarely found in clinical specimens and its pathogenic potential is unclear. The clinical data of prosthetic hip (n = 5) and shoulder (n = 2) joint implant-associated infections where S. saccharolyticus was detected in periprosthetic tissue specimens are described. The prosthetic hip joint infection cases presented as "aseptic" loosening and may represent chronic, insidious, low-grade prosthetic joint infections (PJIs), eventually resulting in loosening of prosthetic components. All cases were subjected to one-stage revision surgery and the long-term outcome was good. The shoulder joint infections had an acute onset. Polymicrobial growth, in all cases with Cutibacterium acnes, was found in 4/7 patients. All but one case were treated with long-term administration of beta-lactam antibiotics. Whole-genome sequencing (WGS) of the isolates was performed and potential virulence traits were identified. WGS could distinguish two phylogenetic clades (clades 1 and 2), which likely represent distinct subspecies of S. saccharolyticus. Little strain individuality was observed among strains from the same clade. Strains of clade 2 were exclusively associated with hip PJIs, whereas clade 1 strains originated from shoulder PJIs. It is possible that strains of the two clades colonize different skin habitats. In conclusion, S. saccharolyticus has the potential to cause PJIs that were previously regarded as aseptic loosening of prosthetic joint devices.

Complete Genome Sequences of Two Staphylococcus saccharolyticus Strains Isolated from Prosthetic Joint Infections.

Staphylococcus saccharolyticus is a human skin bacterium and is occasionally associated with prosthetic joint infections (PJIs). Here, we report the complete genome sequences of two strains that were isolated from shoulder and hip PJIs. The genomes show signs of reductive evolution; around 21% of all coding sequences are inactivated by frameshift mutations.

Clinical and genomic features of Corynebacterium macginleyi-associated infectious keratitis.

Infectious keratitis is a potentially sight threatening ophthalmological emergency. Contact lens wear is a common risk factor. Diagnostic advances such as MALDI-TOF MS provides new insights into the spectrum of corneal pathogens and on microbes previously considered as commensals. Corynebacterium macginleyi was described in 1995, and in 2018, the genomic features of three isolates were reported after whole-genome sequencing. Here we describe the clinical characteristics of patients with infectious keratitis (n = 29) presumably caused by Corynebacterium macginleyi, and analyze the genomic features of C. macginleyi (n = 22) isolated from the corneal ulcers of these patients. The disease course was uneventful apart from minor interventions such as corneal cross-linking and amniotic membrane transplant. Genome sequencing and comparison revealed a highly conserved core genome of C. macginleyi. Based on the analyses of single nucleotide polymorphisms, the population could be divided into two main clades that also differed in a few clade-specific genomic islands. Patients infected with an isolate belonging to the minor clade (n = 7) presented a more severe disease. Comparisons with other corynebacterial species clearly separated C. macginleyi. C. macginleyi may be considered a corneal pathogen; genomic analysis provided insights into its population structure and disease-causing potential.

Staphylococcus saccharolyticus: An Overlooked Human Skin Colonizer.

Coagulase-negative staphylococcal species constitute an important part of the human skin microbiota. In particular, facultative anaerobic species such as Staphylococcus epidermidis and Staphylococcus capitis can be found on the skin of virtually every human being. Here, we applied a culture-independent amplicon sequencing approach to identify staphylococcal species on the skin of healthy human individuals. While S. epidermidis and S. capitis were found as primary residents of back skin, surprisingly, the third most abundant member was Staphylococcus saccharolyticus, a relatively unstudied species. A search of skin metagenomic datasets detected sequences identical to the genome of S. saccharolyticus in diverse skin sites, including the back, forehead, and elbow pit. Although described as a slow-growing anaerobic species, a re-evaluation of its growth behavior showed that S. saccharolyticus can grow under oxic conditions, and, in particular, in a CO2-rich atmosphere. We argue here that S. saccharolyticus was largely overlooked in previous culture-dependent and -independent studies, due to its requirement for fastidious growth conditions and the lack of reference genome sequences, respectively. Future studies are needed to unravel the microbiology and host-interacting properties of S. saccharolyticus and its role as a prevalent skin colonizer.

Dose-Dependent Effects of Common Antibiotics Used to Treat Staphylococcus aureus on Biofilm Formation.

BACKGROUND & OBJECTIVE: Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), represent serious nosocomial and community infections. Biofilm formation as an important virulence factor may be affected by sub-inhibitory levels of antibiotics. Few studies examined the effects of all therapeutic antimicrobial agents on clinical S.aureus. The current study aimed at observing the inducing and reducing effects of antibiotics, commonly used to treat staphylococcal infections on the production of staphylococcal biofilm. METHODS: Four MRSA (1ATCC and 3 clinical) and 1 methicillin-susceptible Staphylococcus aureus (MSSA) strains with biofilm forming ability, evaluated by the Congo red agar (CRA) plate test, were employed. Biofilm formation was measured by crystal violet microtiter plate assay. Cefazolin, rifampicin, vancomycin, oxacillin, clindamycin, cotrimoxazole, minocycline, linezolid, azithromycin, and clarithromycin were added to wells ranging from 0.06to 128 µg/mL (1× to 1/1024 MIC dependent on the MIC value of each strain). RESULTS: The current study showed that azithromycin and vancomycin had a significant inducing effect on biofilm formation. In contrast, linezolid, cefazolin, and clarithromycin, and in the second place, clindamycin and minocycline could inhibit the level of biofilm production in the sub-minimal inhibitory concentrations. CONCLUSION: The findings demonstrated that the biofilm formation as an important virulence factor may be affected by the subinhibitory levels of antibiotics.

Spread of Efflux Pump Overexpressing-Mediated Fluoroquinolone Resistance and Multidrug Resistance in Pseudomonas aeruginosa by using an Efflux Pump Inhibitor.

BACKGROUND: Fluoroquinolone resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression and/or target mutations. We designed this study to investigate the efflux pump mediated fluoroquinolone resistance and check the increasing effectiveness of fluoroquinolones in combination with an efflux pumps inhibitor among P. aeruginosa isolates from burn wounds infections. MATERIALS AND METHODS: A total of 154 consecutive strains of P. aeruginosa were recovered from separate patients hospitalized in a burn hospital, Tehran, Iran. The isolates first were studied by disk diffusion antibiogram for 11 antibiotics and then minimum inhibitory concentration (MIC) experiments were performed to detect synergy between ciprofloxacin and the efflux pump inhibitor, carbonyl cyanide-m-chlorophenyl hydrazone (CCCP). Then to elucidate the inducing of multi drug resistance due to different efflux pumps activation in Fluoroquinolone resistant isolates, synergy experiments were also performed in random ciprofloxacin resistant isolates which have overexpressed efflux pumps phenotypically, using CCCP and selected antibiotics as markers for Beta-lactams and Aminoglycosides. The isolates were also tested by polymerase chain reaction (PCR) for the presence of the MexA, MexC and MexE, which encode the efflux pumps MexAB-OprM, MexCD-OprJ and MexEF-OprN. RESULTS: Most of the isolates were resistant to 3 or more antibiotics tested. More than half of the ciprofloxacin resistant isolates exhibited synergy between ciprofloxacin and CCCP, indicating the efflux pump activity contributed to the ciprofloxacin resistance. Also increased susceptibility of random ciprofloxacin resistant isolates of P. aeruginosa to other selected antibiotics, in presence of CCCP, implied multidrug extrusion by different active efflux pump in fluoroquinolones resistant strains. All of Ciprofloxacin resistant isolates were positive for MexA, MexC and MexE genes simultaneously. CONCLUSION: In this burn hospital, where multidrug resistant P. aeruginosa isolates were prevalent, ciprofloxacin resistance and multidrug resistance due to the overexpression of fluoroquinolones mediated efflux pumps has also now emerged. Early recognition of this resistance mechanism should allow the use of alternative antibiotics and use an efflux pumps inhibitor in combination with antibiotic therapy.

Characterisation of SCCmec elements in methicillin-resistant Staphylococcus aureus isolated from burn patients.

Staphylococcus aureus is an important pathogen, especially in burn units all around the world. Because of the emergence of the ß-lactam antibiotic-resistant strains since 1961, concern about the prevalence of methicillin-resistant S. aureus (MRSA) has increased in these units. Resistance to methicillin is mediated by penicillin-binding proteins (PBPs) that have enough affinity for binding to the ß-lactam ring, but another kind of protein (PBP2α), which is encoded by the mecA gene, has a lower affinity for binding to these antibiotics. The mecA gene is transferred by SCCmec (staphylococcal cassette chromosome mec) as a mobile genetic element, exclusively found in the Staphylococcus genus. Identification of the frequency of the mecA gene, different SCCmec types and also its incidence may have benefit in surveillance prevention and control of MRSA strains in burn units. In this study, 40 S. aureus isolates were collected from patients hospitalised in Motahari burn center of Tehran, during 2012-2013. Conventional microbiological methods were applied and the confirmed isolates were stored at -20°C for molecular polymerase chain reaction (PCR) tests. The antibiotic resistance pattern was performed by disc diffusion method and finally the different SCCmec types were determined by specific primers. During this research, 40 isolates of S. aureus were collected from burn patients, of which (37.5%) of the specimens belonged to female patients and 62.5% to male patients. The aetiology of the burn was classified as follows: open flame (35%), liquid (32.5%), chemical (5%) and other (27.5%). By a disc diffusion method, no resistance pattern was observed to vancomycin and fosfomycin. Based on a multiplex PCR assay, the five different SCCmec types were detected as: 47.5% type III, 25% type IV, 10% type V, 10% type II and 7.5% type I.