OBJECTIVE: To explore the mechanism of antibiotic delivery system targeting bacterial biofilm with linezolid (LZD) based on ε-poly- L-lysine (ε-PLL) and cyclodextrin (CD) (ε-PLL-CD-LZD), aiming to enhance antibiotic bioavailability, effectively penetrate and disrupt biofilm structures, and thereby improve the treatment of bone and joint infections. METHODS: ε-PLL-CD-LZD was synthesized via chemical methods. The grafting rate of CD was characterized using nuclear magnetic resonance. In vitro biocompatibility was evaluated through live/dead cell staining after co-culturing with mouse embryonic osteoblast precursor cells (MC3T3-E1), human umbilical vein endothelial cells, and mouse embryonic fibroblast cells (3T3-L1). The biofilm-enrichment capacity of ε-PLL-CD-LZD was assessed using Staphylococcus aureus biofilms through enrichment studies. Its biofilm eradication efficacy was investigated via minimum inhibitory concentration (MIC) determination, scanning electron microscopy, and live/dead bacterial staining. A bone and joint infection model in male Sprague-Dawley rats was established to validate the antibacterial effects of ε-PLL-CD-LZD. RESULTS: In ε-PLL-CD-LZD, the average grafting rate of CD reached 9.88%. The cell viability exceeded 90% after co-culturing with three types cells. The strong biofilm enrichment capability was observed with a MIC of 2 mg/L. Scanning electron microscopy observations revealed the effective disruption of biofilm structure, indicating potent biofilm eradication capacity. In vivo rat experiments demonstrated that ε-PLL-CD-LZD significantly reduced bacterial load and infection positivity rate at the lesion site ( P<0.05). CONCLUSION The ε-PLL-CD antibiotic delivery system provides a treatment strategy for bone and joint infections with high clinical translational significance. By effectively enhancing antibiotic bioavailability, penetrating, and disrupting biofilms, it demonstrated significant anti-infection effects in animal models.
Biofilms/drug effects* ; Animals ; Anti-Bacterial Agents/pharmacology* ; Polylysine/chemistry* ; Cyclodextrins/administration & dosage* ; Humans ; Linezolid/pharmacology* ; Staphylococcus aureus/physiology* ; Rats, Sprague-Dawley ; Mice ; Rats ; Male ; Drug Delivery Systems ; Staphylococcal Infections/drug therapy* ; Microbial Sensitivity Tests ; Human Umbilical Vein Endothelial Cells ; Osteoblasts/cytology*

Humans ; Hydrogen-Ion Concentration ; Malassezia ; growth & development ; physiology ; Staphylococcus epidermidis ; growth & development ; physiology

BACKGROUNDSepsis is one of the main causes of mortality in critically ill patients following progression to septic shock. To investigate the pathophysiologic changes of sepsis, we developed a novel porcine model of septic shock induced by acute respiratory distress syndrome (ARDS) due to methicillin-resistant Staphylococcus aureus(MRSA) pneumonia.

METHODSTwenty-six male Landraces (Lvyuanweiye, Beijing, China) weighing 30 ± 2 kg were divided into four groups: sham group (SH; n = 5); cotton smoke inhalation group (SM; n = 6); MRSA pneumonia group (MR; n = 6); and septic shock group with cotton smoke inhalation + MRSA pneumonia (SS; n = 9). Extensive hemodynamics, oxygen dynamics, and lung function were monitored for 24 h following the injury or until death. Tissues were collected, and histopathology evaluations were carried out.

RESULTSBlood cultures from 6 of 9 animals in the SS group were positive for MRSA. Two hours following the injury, decreased mean arterial blood pressure (60-70 mmHg) and cardiac index (<2 L.min-1.m-2) were observed in the animals in the SS group, while systemic vascular resistance index was increased. The hemodynamic characteristics of septic shock were only observed in the SS group but not significant in the other groups. The PO2/FiO2in the SM and SS groups decreased to 300 and 100, respectively. In the SS group, extravascular lung water index increased to 20 ml/kg, whereas thoracopulmonary compliance decreased to 10 ml/H2O after injury. Deterioration of pulmonary function in the SS group was more serious than the SM and MR groups. Severe lung injury in the SS group was confirmed by the histopathology evaluations. The lung injury confirmed by high-resolution thin-section computed tomography and histopathology in the SS group was more serious than those of other groups.

CONCLUSIONSIn the present study, we developed a novel porcine model of septic shock induced by ARDS due to severe MRSA pneumonia with characteristic hyperdynamic and hypodynamic phases in 24 h, which mimicked the hemodynamic changing of septic shock in human.

Animals ; Disease Models, Animal ; Hemodynamics ; physiology ; Male ; Methicillin-Resistant Staphylococcus aureus ; pathogenicity ; Pneumonia ; microbiology ; pathology ; Respiratory Distress Syndrome, Adult ; complications ; pathology ; Shock, Septic ; etiology ; pathology ; Swine

Three new alkyl substituted anthraquinone derivatives, trivially named as symploquinones A-C (Compounds 1-3) were isolated from Symplocos racemosa. The structures of these compounds were determined on the basis of extensive spectroscopic analyses (UV, IR, Mass, H- and C-NMR, and two-dimensional (2D) NMR techniques). The resulting data were also compared with the reported literature. These compounds were then subjected to antibacterial or antibiofilm testing. Compounds 1 and 3 exhibited good antibacterial activity in the concentration range of 160-83 μg·mL against Streptococcus mutans, methicillin resistant Staphylococcus aureus and Proteus mirabilis. Both compounds were further screened for anti-biofilm activity, which revealed promising activities at sub-MIC concentrations. None of the compounds were found to be active against Klebsiella pneumoniae.
Anthraquinones ; chemistry ; isolation & purification ; pharmacology ; Anti-Bacterial Agents ; chemistry ; isolation & purification ; pharmacology ; Biofilms ; drug effects ; growth & development ; Ericales ; chemistry ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; physiology ; Microbial Sensitivity Tests ; Proteus mirabilis ; drug effects ; physiology ; Spectrophotometry, Infrared ; Streptococcus mutans ; drug effects ; physiology

Amphibian skin contains rich bioactive peptides. Especially, a large amount of antimicrobial peptides have been identified from amphibian skin secretions. Antimicrobial peptides display potent cytolytic activities against a range of pathogenic bacteria and fungi and play important defense roles. No antimicrobial peptides have been reported from toads belonging to the family of Pelobatidae. In this work, two novel antimicrobial peptides (Megin 1 and Megin 2) were purified and characterized from the skin venoms of spadefoot toad Megophrys minor (Pelobatidae, Anura, Amphibia). Megin 1 had an amino acid sequence of FLKGCWTKWYSLKPKCPF-NH2, which was composed of 18 amino acid residues and contained an intra-molecular disulfide bridge and an amidated C-terminus. Megin 2 had an amino acid sequence of FFVLKFLLKWAGKVGLEHLACKFKNWC, which was composed of 27 amino acid residues and contained an intra-molecular disulfide bridge. Both Megin 1 and Megin 2 showed potential antimicrobial abilities against bacteria and fungi. The MICs of Megin 1 against Escherichia coli, Bacillus dysenteriae, Staphylococcus aureus, Bacillus subtilis, and Candida albicans were 25, 3, 6.25, 3, and 50 μg·mL(-1), respectively. The corresponding MICs for Megin 2 were 6.25, 1.5, 12.5, 1.5, and 12.5 μg·mL(-1), respectively. They also exerted strong hemolytic activity against human and rabbit red cells. The results suggested that megin peptides in the toad skin of M. minor displayed toxic effects on both eukaryotes and prokaryotes. This was the first report of antimicrobial peptides from amphibians belonging to the family of Pelobatidae.
Amino Acid Sequence ; Amphibian Venoms ; chemistry ; immunology ; isolation & purification ; Animals ; Anura ; immunology ; Bacillus ; Candida albicans ; Erythrocytes ; physiology ; Escherichia coli ; Female ; Hemolysis ; Humans ; Male ; Peptides ; chemistry ; immunology ; isolation & purification ; Rabbits ; Sequence Alignment ; Skin ; chemistry ; immunology ; Staphylococcus aureus

OBJECTIVETo study the effects of inhibitory peptide of Staphylococcus epidermidis (SE) biofilm (briefly referred to as inhibitory peptide) on adhesion and biofilm formation of SE at early stage.

METHODSBy using peptide synthesizer, the inhibitory peptide was synthesized with purity of 96.8% and relative molecular mass of 874.4. (1) Solution of SE ATCC 35984 (the same below) was cultivated with inhibitory peptide in the final concentrations of 1-256 µg/mL, and the M-H broth without bacteria solution was used as blank control. The MIC of the inhibitory peptide against SE was determined (n=3). (2) Solution of SE was cultivated with trypticase soy broth (TSB) culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Growth of SE was observed every one hour from immediately after cultivation (denoted as absorbance value), and the growth curve of SE during the 24 hours of cultivation was drawn, with 3 samples in each group at each time point. (3) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE was observed after cultivation for 4 hours (denoted as absorbance value, n=10); biofilm formation of SE was observed after cultivation for 20 hours (denoted as absorbance value, n=10). (4) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentration of 128 µg/mL (set as 128 µg/mL inhibitory peptide group), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE and its biofilm formation were observed with confocal laser scanning microscope (CLSM), and the sample numbers were both 3. Data were processed with one-way analysis of variance, LSD test, and Dunnett T3 test.

RESULTS(1) The MIC of inhibitory peptide against SE exceeded 256 µg/mL. (2) There was no significant difference in the growth curve of SE between inhibitory peptide groups in different concentrations and negative control group. (3) After 4 hours of cultivation, the absorbance values of adhesive property of SE in 256, 128, 64, and 32 µg/mL inhibitory peptide groups were respectively 0.20 ± 0.04, 0.27 ± 0.03, 0.35 ± 0.04, and 0.40 ± 0.04, which were significantly lower than the absorbance value in negative control group (0.53 ± 0.10, P<0.05 or P<0.01); the absorbance value of adhesive property of SE in 16 µg/mL inhibitory peptide group was 0.47 ± 0.09, which was close to the absorbance value in negative control group (P>0.05). After 20 hours of cultivation, the absorbance values of biofilm formation of SE in 256, 128, and 64 µg/mL inhibitory peptide groups were respectively 0.49 ± 0.10, 0.68 ± 0.06, and 0.93 ± 0.13, which were significantly less than the absorbance value in negative control group (1.21 ± 0.18, P<0.05 or P<0.01); the absorbance values of biofilm formation in 32 and 16 µg/mL inhibitory peptide groups were respectively 1.18 ± 0.22 and 1.15 ± 0.26, which were close to the absorbance value in negative control group (with P values above 0.05). (4) CLSM showed that more adhering bacteria and compact structure of biofilm were observed in negative control group, but less adhering bacteria and loose structure of biofilm were observed in 128 µg/mL inhibitory peptide group.

CONCLUSIONSThe inhibitory peptide can inhibit adhesion and biofilm formation of SE at early stage, but its structure still needs to be further modified.

Bacterial Adhesion ; Biofilms ; growth & development ; Humans ; Microscopy, Confocal ; Peptides ; Staphylococcus epidermidis ; genetics ; metabolism ; physiology

BACKGROUNDDiversity of orthopedic infections with various local environments affects the pattern and prevalence of pathogens. It is not well-characterized whether different pathogens have different propensity to cause different types of orthopedic infections. We aimed to investigate the frequency of different pathogens derived from orthopedic infections, and determine the relationship between the prevalence of clinical isolates and the type of orthopedic implants, especially focusing on staphylococci.

METHODSFrom January 2006 to December 2011, orthopedic infections were identified retrospectively from clinical microbiology laboratory and orthopedic medical records. The sources of orthopedic infections were divided into two main groups: those associated with implants and those not associated with implants. Implants-associated infections were further subdivided into five subgroups: arthroplasty, internal fixation, external fixation, internal and external fixation, and others. We analyzed microbiological spectrum in different groups and subgroups. Antibiotic susceptibility of staphylococci was analyzed.

RESULTSOnly coagulase-negative staphylococci (CoNS) was significantly more likely to be associated with implants-associated infections (P = 0.029). The overall pathogens prevalence of arthroplasty was significantly different from other subgroups (P < 0.05). 65% isolates from external fixation was Gram-negative bacteria. Some percentage (55%) of S. aureus and (83%) CoNS were resistant to methicillin. No resistance to glycopeptide was seen in all of staphylococci.

CONCLUSIONSStaphylococcus aureus was the most frequent isolates in orthopedic infections but was not associated with the presence or absence of implants. Only CoNS was implants-associated, especially for arthroplasty infection. Cefazolin alone is not enough for orthopedic surgery prophylaxis in settings with a high prevalence of methicillin-resistant staphylococci.

Humans ; Orthopedic Procedures ; adverse effects ; Prostheses and Implants ; microbiology ; Retrospective Studies ; Staphylococcus aureus ; pathogenicity ; physiology

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.
Animals ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Viral/physiology ; Mucoproteins/genetics/*metabolism ; Phylogeny ; Polymerase Chain Reaction/methods ; Recombinant Proteins ; Staphylococcus Phages/genetics/*metabolism/physiology ; Staphylococcus aureus/*virology

OBJECTIVETo observe the effect of the combination of sub-MIC sodium houttuyfonate and erythromycin on biofilm of Staphylococcus epidermidis.

METHODThe serial dilution method was adopted to determine MIC of the combination of sodium houttuyfonate and erythromycin on S. epidermidis; the checkerboard method was used to evaluate the combination of sodium houttuyfonate and erythromycin on suspended bacteria of S. epidermidis; S. epidermidis biofilm was built in vitro, and XTT reduction assay was used to evaluate the effect of the combination of sub-MIC sodium houttuyfonate and erythromycin on the adhesion of S. epidermidis and bacterial metabolism inside the biofilm. Microscope was applied to observe the impact the single administration and combination of the two medicines under sub-MIC on biofilm morphology of S. epidermidis.

RESULTThe MIC of sodium houttuyfonate and erythromycin were 62.5, 7.812 5 mg x L(-1), respectively. The combination of 1/8MIC sodium houttuyfonate and 1/2MIC erythronmycin showed a synergistic effect on S. epidermidis. Sodium houttuyfonate, erythromycin and their combination had an inhibitory effect on the adhesion and metabolism of S. epidermidis biofilm bacteria, and made impact on the morphology of S. epidermidis biofilm.

CONCLUSIONThe sub-MIC sodium houttuyfonate and erythromycin have an inhibitory effect on S. epidermidis biofilm. The combination of sodium houttuyfonate and erythromycin shows a synergistic effect in inhibiting suspended bacteria and biofilm of S. epidermidis, particularly in inhibiting the metabolism of S. epidermidis biofilm bacteria and impacting the morphology of biofilm.

Alkanes ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Bacterial Adhesion ; drug effects ; Biofilms ; drug effects ; growth & development ; Dose-Response Relationship, Drug ; Drug Interactions ; Erythromycin ; pharmacology ; Microbial Sensitivity Tests ; Staphylococcus epidermidis ; drug effects ; physiology ; Sulfites ; pharmacology

To assess the ability of the previously selected human vaginal isolates of Lactobacillus crispatus (L. crispatus) T79-3, T90-1 and Lactobacillus jensenii (L. jensenii) T118-3, T231-1 to inhibit the growth of Staphylococcus aureus and block their adhesion to HeLa cells. The inhibitory bioactive substances produced by these Lactobacillus were also identified. Inhibitory substances interaction tests were carried out by using a streak-diffusion method on agar plates. Three types of interaction were performed to determine the inhibitory effect of Lactobacillus on adhesion of Staphylococcus aureus to HeLa cells: Exclusion Group (Lactobacillus and HeLa followed by pathogens), Competition Group (Lactobacillus, HeLa and pathogens together) and Displacement Group (pathogens and HeLa followed by the addition of Lactobacillus). The number of HeLa cells adhered to Staphylococcus aureus was quantified by bacteria colony counts on LB plate. The results showed that lactic acids produced by the Lactobacillus are the main substances that can inhibit Staphylococcus aureus growth and there is variation among the three types of interaction regarding the inhibitory activity against Staphylococcus aureus. The effects of Lactobacillus on blocking the adhesion to HeLa cells were concentration dependent. All four Lactobacillus isolates displayed the ability to inhibit Staphylococcus aureus growth and block Staphylococcus aureus adherence to HeLa cells. Exclusion Group was the most effective, and T79-3 showed greater capacity to block Staphylococcus aureus adherence compared with the other three isolates. The present study suggests the potential ability of L. crispatus T79-3 as probiotic for the treatment and prevention of urogenital infections in women.
Bacterial Adhesion ; physiology ; Cell Wall ; chemistry ; Female ; HeLa Cells ; Humans ; Lactobacillus ; classification ; physiology ; Probiotics ; Staphylococcus aureus ; growth & development ; pathogenicity ; Vagina ; microbiology