Can platelet-rich fibrin act as a natural carrier for antibiotics delivery? A proof-of-concept study for oral surgical procedures.

OBJECTIVES: Evaluate the role of platelet-rich fibrin (PRF) as a natural carrier for antibiotics delivery through the analysis of drug release and antimicrobial activity. MATERIALS AND METHODS: PRF was prepared according to the L-PRF (leukocyte- and platelet-rich fibrin) protocol. One tube was used as control (without drug), while an increasing amount of gentamicin (0.25 mg, G1; 0.5 mg, G2; 0.75 mg, G3; 1 mg, G4), linezolid (0.5 mg, L1; 1 mg, L2; 1.5 mg, L3; 2 mg, L4), vancomycin (1.25 mg, V1; 2.5 mg, V2; 3.75 mg, V3; 5 mg, V4) was added to the other tubes. At different times the supernatant was collected and analyzed. Strains of E. coli, P. aeruginosa, S. mitis, H. influenzae, S. pneumoniae, S. aureus were used to assess the antimicrobial effect of PRF membranes prepared with the same antibiotics and compared to control PRF. RESULTS: Vancomycin interfered with PRF formation. Gentamicin and linezolid did not change the physical properties of PRF and were released from membranes in the time intervals examined. The inhibition area analysis showed that control PRF had slight antibacterial activity against all tested microorganisms. Gentamicin-PRF had a massive antibacterial activity against all tested microorganisms. Results were similar for linezolid-PRF, except for its antibacterial activity against E. coli and P. aeruginosa that was comparable to control PRF. CONCLUSIONS: PRF loaded with antibiotics allowed the release of antimicrobial drugs in an effective concentration. Using PRF loaded with antibiotics after oral surgery may reduce the risk of post-operative infection, replace or enhance systemic antibiotic therapy while preserving the healing properties of PRF. Further studies are needed to prove that PRF loaded with antibiotics represents a topical antibiotic delivery tool for oral surgical procedures.

Antibiotic-Lysobacter enzymogenes proteases combination as a novel virulence attenuating therapy.

Minimizing antibiotic resistance is a key motivation strategy in designing and developing new and combination therapy. In this study, a combination of the antibiotics (cefixime, levofloxacin and gentamicin) with Lysobacter enzymogenes (L. enzymogenes) bioactive proteases present in the cell- free supernatant (CFS) have been investigated against the Gram-positive methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative Escherichia coli (E. coli O157:H7). Results indicated that L. enzymogenes CFS had maximum proteolytic activity after 11 days of incubation and higher growth inhibitory properties against MSSA and MRSA compared to E. coli (O157:H7). The combination of L. enzymogenes CFS with cefixime, gentamicin and levofloxacin at sub-MIC levels, has potentiated their bacterial inhibition capacity. Interestingly, combining cefixime with L. enzymogenes CFS restored its antibacterial activity against MRSA. The MTT assay revealed that L. enzymogenes CFS has no significant reduction in human normal skin fibroblast (CCD-1064SK) cell viability. In conclusion, L. enzymogenes bioactive proteases are natural potentiators for antimicrobials with different bacterial targets including cefixime, gentamicin and levofloxacin representing the beginning of a modern and efficient era in the battle against multidrug-resistant pathogens.

Enhancement of Inhibition of the Pseudomonas sp. Biofilm Formation on Bacterial Cellulose-Based Wound Dressing by the Combined Action of Alginate Lyase and Gentamicin.

Bacterial biofilms generally contribute to chronic infections, including wound infections. Due to the antibiotic resistance mechanisms protecting bacteria living in the biofilm, they are a serious problem in the wound healing process. To accelerate the wound healing process and avoid bacterial infection, it is necessary to select the appropriate dressing material. In this study, the promising therapeutic properties of alginate lyase (AlgL) immobilised on BC membranes for protecting wounds from Pseudomonas aeruginosa infection were investigated. The AlgL was immobilised on never dried BC pellicles via physical adsorption. The maximum adsorption capacity of AlgL was 6.0 mg/g of dry BC, and the equilibrium was reached after 2 h. The adsorption kinetics was studied, and it has been proven that the adsorption was consistent with Langmuir isotherm. In addition, the impact of enzyme immobilisation on bacterial biofilm stability and the effect of simultaneous immobilisation of AlgL and gentamicin on the viability of bacterial cells was investigated. The obtained results showed that the AlgL immobilisation significantly reduced the amount of polysaccharides component of the P. aeruginosa biofilm. Moreover, the biofilm disruption by AlgL immobilised on BC membranes exhibited synergism with the gentamicin, resulting in 86.5% more dead P. aeruginosa PAO-1 cells.

Protective Effect of Neutral Electrolyzed Saline on Gentamicin-Induced Nephrotoxicity: Evaluation of Histopathologic Parameters in a Murine Model.

Background and Objectives: Gentamicin (GM) is a nephrotoxic aminoglycoside. Neutral electrolyzed saline (SES) is a compound with anti-inflammatory, antioxidant, and immunomodulatory properties. The objective of the present study was to evaluate whether kidney damage by GM can be prevented and/or reversed through the administration of SES. Materials and Methods: The study was carried out as a prospective, single-blind, five-arm, parallel-group, randomized, preclinical trial. The nephrotoxicity model was established in male BALB/c mice by administering GM at a dose of 100 mg/kg/day intraperitoneally for 30 days, concomitantly administering (+) SES or placebo (physiologic saline solution), and then administering SES for another 30 days after the initial 30 days of GM plus SES or placebo. At the end of the test, the mice were euthanized, and renal tissues were evaluated histopathologically. Results: The GM + placebo group showed significant tubular injury, interstitial fibrosis, and increased interstitial infiltrate of inflammatory cells compared with the group without GM. Tubular injury and interstitial fibrosis were lower in the groups that received concomitant GM + SES compared with the GM + placebo group. SES administration for 30 days after the GM administration periods (GM + placebo and GM + SES for 30 days) did not reduce nephrotoxicity. Conclusions: Intraperitoneal administration of SES prevents gentamicin-induced histologic nephrotoxicity when administered concomitantly, but it cannot reverse the damage when administered later.

Formation of Listeria monocytogenes persister cells in the produce-processing environment.

Persisters are a subpopulation of growth-arrested cells that possess transient tolerance to lethal doses of antibiotics and can revert to an active state under the right conditions. Persister cells are considered as a public health concern. This study examined the formation of persisters by Listeria monocytogenes (LM) in an environment simulating a processing plant for leafy green production. Three LM strains isolated from California produce-processing plants and packinghouses with the strongest adherence abilities were used for this study. The impact of the cells' physiological status, density, and nutrient availability on the formation of persisters was also determined. Gentamicin at a dose of 100 mg/L was used for the isolation and screening of LM persisters. Results showed that the physiological status differences brought by culture preparation methods (plate-grown vs. broth-grown) did not impact persister formation (P > 0.05). Instead, higher persister ratios were found when cell density increased (P < 0.05). The formation of LM persister cells under simulated packinghouse conditions was tested by artificially inoculating stainless steel coupons with LM suspending in media with decreasing nutrient levels: brain heart infusion broth (1366 mg/L O2), produce-washing water with various organic loads (1332 mg/L O2 and 652 mg/L O2, respectively), as well as sterile Milli-Q water. LM survived in all suspensions at 4 °C with 85 % relative humidity for 7 days, with strain 483 producing the most persister cells (4.36 ± 0.294 Log CFU/coupon) on average. Although persister cell levels of LM 480 and 485 were reasonably steady in nutrient-rich media (i.e., BHI and HCOD), they declined in nutrient-poor media (i.e., LCOD and sterile Milli-Q water) over time. Persister populations decreased along with total viable cells, demonstrating the impact of available nutrients on the formation of persisters. The chlorine sensitivity of LM persister cells was evaluated and compared with regular LM cells. Results showed that, despite their increased tolerance to the antibiotic gentamicin, LM persisters were more susceptible to chlorine treatments (100 mg/L for 2 min) than regular cells.

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus.

Background: Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. Aim: In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Methods: Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. Results: The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. Conclusion: This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.

Functionalization of ampicillin and gentamicin with biogenic copper nanoparticles (CuNPs) remodel antimicrobial and cytotoxic outcome against MDR clinical isolates.

The present study reports the functionalization of antibiotic-conjugated Alternanthera pungens and Trichodesma indicum copper nanoparticles (CuNPs). Initially, antibiotic profiling of multi-drug resistant (MDR) clinical isolates against five antibiotics was verified and then gentamicin and ampicillin conjugates of CuNPs were prepared. Biosynthesized nanostructures were characterized through UV-visible spectroscopy, Fourier-transformed infrared spectroscopy, X-ray diffraction and scanning electron microscope. Biogenic synthesized CuNPs displayed highest antibacterial activity (24.0-31.3 mm inhibition zones) when capped with gentamicin as compared to the ampicillin-conjugated NPs which showed resistance against most of the bacterial species. A. pungens-derived conjugates of gentamicin (CuAp-GNT) along with the vehicle revealed 4.86 ± 0.20% and 4.25 ± 2.96% hemolytic potential and highest MDA production in S. typhimurium (3.18 ± 1.52 µg/mL and 6.31 ± 3.49 µg/mL) and K. pneumoniae (2.99 ± 0.90 µg/mL and 4.06 ± 1.20 µg/mL). Similarly, CuAp-GNT also showed highest DNA protection ability by displaying 1342.99 ± 11.87 band intensity. All-inclusive, CuAp showed more promising effects when conjugated with gentamicin indicating that capping of gentamicin with the active components of the plant-based copper nanostructures increases the antibacterial capacity of the drug. Hence, conjugation of antibiotics with bio-based sources offers great potential for identifying potent drug leads.

Prevalence of Class 1 Integron and In Vitro Effect of Antibiotic Combinations of Multidrug-Resistant Enterococcus Species Recovered from the Aquatic Environment in the Eastern Cape Province, South Africa.

Enterococci are regarded as a better indication of faecal pollution in freshwater and marine waters. Their levels in seawater are positively connected with swimming-related gastrointestinal disorders. This study used an Enterococcus-specific polymerase chain reaction (PCR) to characterize the isolates. Classes 1 and 2 integrons were examined for environmental Enterococcus isolates using a standard biological procedure. All strains were assessed against a panel of 12 antibiotics from various classes using disc diffusion methods. The microdilution method was used to work out the minimum inhibitory concentration (MIC) according to the CLSI guiding principles. The combination therapy of the resistant drugs was evaluated using a checkerboard assay and a time-dependent test for assessing their bactericidal or bacteriostatic activity. The gene diversity of the tested organisms was analyzed with the aid of Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR. In total, 57 Enterococcus spp. environmental samples were recovered, in which Enterococcus faecalis (33.33%) and Enterococcus faecium (59.65%) were the dominant species. Resistance to linezolid, ciprofloxacin, erythromycin, gentamicin, vancomycin, rifampicin, and tetracycline was prevalent. Fifty (50) strains tested positive for class 1 integron, more frequent in Enterococcus faecium and Enterococcus faecalis isolates, with no gene cassette array discovered. A combination of gentamicin (MIC 4 µg/mL) with vancomycin (MIC 256 µg/mL) antibiotics against Enterococcus faecalis showed antibacterial activity. In contrast, the combination of ciprofloxacin (1 µg/mL) with Ampicillin (16 µg/mL) antibiotics against Enterococcus faecalis showed a bacteriostatic effect. The ERIC-PCR analysis pointed out that most of the assessed isolates have close genetic similarities.

Quantitative proteomics reveals reduction in central carbon and energy metabolisms contributes to gentamicin resistance in Staphylococcus aureus.

The emergence of antibiotic resistance greatly increases the difficulty of treating bacterial infections. In order to develop effective treatments, the underlying mechanisms of antibiotic resistance must be understood. In this study, Staphylococcus aureus ATCC6538 strain was passaged in medium with and without gentamicin and obtained lab-evolved gentamicin-resistant S. aureus (RGEN) and gentamicin-sensitive S. aureus (SGEN) strains, respectively. Data-Independent Acquisition (DIA)-based proteomics approach was applied to compare the two strains. A total of 1426 proteins were identified, of which 462 were significantly different: 126 were upregulated and 336 were downregulated in RGEN compared to SGEN. Further analysis found that reduced protein biosynthesis was a characteristic feature in RGEN, related to metabolic suppression. The most differentially expressed proteins were involved in metabolic pathways. In RGEN, central carbon metabolism was dysregulated and energy metabolism decreased. After verification, it was found that the levels of NADH, ATP, and reactive oxygen species (ROS) decreased, and superoxide dismutase and catalase activities increased. These findings suggest that inhibition of central carbon and energy metabolic pathways may play an important role in the resistance of S. aureus to gentamicin, and that gentamicin resistance is associated with oxidative stress. Significance: The overuse and misuse of antibiotics have led to bacterial antibiotic resistance, which is a serious threat to human health. Understanding the mechanisms of antibiotic resistance will help better control these antibiotic-resistant pathogens in the future. The present study characterized the differential proteome of gentamicin-resistant Staphylococcus aureus using the most advanced DIA-based proteomics technology. Many of the differential expressed proteins were related to metabolism, specifically, reduced central carbon and energy metabolism. Lower levels of NADH, ROS, and ATP were detected as a consequence of the reduced metabolism. These results reveal that downregulation of protein expression affecting central carbon and energy metabolisms may play an important role in the resistance of S. aureus to gentamicin.

Mitochondrial transplantation against gentamicin-induced toxicity on rat renal proximal tubular cells: the higher activity of female rat mitochondria.

Mitochondrial dysfunction is a fundamental mechanism leading to drug nephrotoxicity, such as gentamicin-induced nephrotoxicity. Mitochondrial therapy (mitotherapy) or exogenous mitochondria transplantation is a method that can be used to replace dysfunctional mitochondria with healthy mitochondria. This method can help in the treatment of diseases related to mitochondria. In this research, we studied the transplantation effect of freshly isolated mitochondria on the toxicity induced by gentamicin on renal proximal tubular cells (RPTCs). Furthermore, possible gender-related effects on supplying exogenous rat kidney mitochondria on gentamicin-induced RPTCs were investigated. At first, the normality and proper functioning of fresh mitochondria were assessed by measuring mitochondrial succinate dehydrogenase activity (SDH) and changes in mitochondrial membrane potential (MMP). Then, the protective effects of mitochondrial transplantation against gentamicin-induced mitochondrial toxicity were evaluated through parameters including lactate dehydrogenase (LDH) leakiness, reactive oxygen species (ROS) production, lipid peroxidation (LPO) content, reduced glutathione (GSH) level, extracellular oxidized glutathione (GSSG) level, ATP level, MMP collapse, and caspase-3 activity. According to the statistical analysis, transplanting the healthy mitochondria decreased the cytotoxicity, ROS production, MMP collapse, LPO content, GSSG levels, and caspase-3 activity caused by gentamicin in RPTCs. Also, it has caused an increase in the level of ATP and GSH in the RPTCs. Furthermore, higher preventive effects were observed for the female group. According to the current study, mitochondrial transplantation is a potent therapeutic method in xenobiotic-caused nephrotoxicity.

In vitro and Ex vivo Assessments of the Compatibility of Fibrin Sealant with Antimicrobial Compounds.

Background: Fibrin sealants are used as antimicrobial-releasing carriers for preventing surgical site infections; however, it is important to determine the release kinetics and antimicrobial effects of drugs added to fibrin sealants and the effects of drugs on clot/clotting properties. Materials and Methods: The antimicrobial and antibiofilm activity of cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate released from fibrin sealant were characterized using in vitro and ex vivo assays against bacteria commonly found on the skin. The effects of antimicrobial agents on the physical structure of the fibrin sealant were assessed with scanning electron microscopy (SEM) and on the clotting rate and strength of fibrin clots using run-off tests and rheology. Results: Generally, antibiotic agents were released gradually from fibrin sealant and were stable after release, with antimicrobial effects evident up to three days. Cefazolin, gentamicin, and oxacillin prevented biofilm formation of Staphylococcus aureus in porcine skin explants; gentamicin and colistin prevented biofilm formation of Pseudomonas aeruginosa. Gentamicin, cefazolin, colistin, and tobramycin did not affect the structural integrity or viscoelastic properties of fibrin sealant; changes were observed with oxacillin (SEM) and particularly silver nitrate (SEM and rheology). No antimicrobial agents caused deterioration of clotting time (run-off tests). Conclusions: From the antimicrobial agents tested, gentamicin and cefazolin showed prolonged release from fibrin sealant, sustained antimicrobial activity, and biofilm prevention properties against Staphylococcus aureus; similar results were observed for gentamicin and colistin against Pseudomonas aeruginosa. For each of these findings, the physical structure of the fibrin sealant, clotting rate, and strength of fibrin clots were unaffected.

Dual drug-loaded hydrogels with pH-responsive and antibacterial activity for skin wound dressing.

Antibacterial and hemostatic properties are essential for wound healing dressing. In this study, a new type of hydrogel composed of gelatin methacryloyl (GelMA) and hyaluronic acid-aldehyde (HA-CHO) is fabricated by photo-crosslinking and respectively loaded with a single drug gentamicin sulfate (GS), and two drugs of GS and lysozyme (LZM). The composite hydrogel of GelMA and HA-CHO is successfully synthesized by the aldehyde and Schiff base reactions. The structures and compositions of the hydrogels with and without drug loaded are characterized by FT-IR, 1H NMR, and XPS. Furthermore, the microstructure and swelling behaviour of hydrogels prove that the content of HA-CHO has a significant role in the formation of hydrogels with dense porous structures and super absorbent. pH 7.4 and pH 5.0 conditions are used to evaluate the drug release behaviour of the obtained hydrogels. The released amount of GS of the drug-loaded hydrogels in the acidic buffer is more than that of the physiological environment because of the cleaved Schiff base bonds and the electrostatic interaction. Especially for the dual drug-loaded hydrogel GelMA/HA-CHO/GS/LZM, the released ratio of GS is elevated from 59 % in pH 7.4 buffer to about 78 % in pH 5.0 buffer within the first 6 h, which verifies the excellent pH-stimulus responsiveness. These endow the GS-LZM dual drug-loaded hydrogels with superior antibacterial efficiencies to that of the single GS drug-loaded hydrogels, no drug-loaded hydrogels, and SEBS control, especially in inhibiting S. aureus in a lower concentration of 106 CFU mL-1, which can be attributed to the synergistic effect of LZM and GS. For S. aureus at 106 CFU mL-1, the bacterial survival of GelMA/HA-CHO/GS/LZM is 1.1 %, which shows outstanding antibacterial effect. Hence, the drug-loaded hydrogels, especially the dual drug-loaded hydrogels with pH-responsive, antibacterial, and hemostatic properties have great potential as wound healing materials.

Genomics and structural insight into the masking of gentamicin-resistance in clinical Burkholderia pseudomallei strain VB29710 from India.

The present study reported a rare gentamicin-susceptible ß-lactamase (PenA, OXA-57) expressing clinical Burkholderia pseudomallei isolate VB29710 from India. Whole-genome sequencing and structural analyses revealed the insertion of R962 and L963 into AmrB, the transmembrane-protein of the AmrAB-OprA efflux-pump that affected aminoglycoside-efflux through local alterations in backbone conformation.

Carboxymethyl cellulose based sustainable hydrogel for colon-specific delivery of gentamicin.

The current research includes the synthesis, improvement of NaCMC-cl-DMAA/AAc hydrogel and in-situ controlled release of gentamicin within various pH environments. The prepared hydrogel was then modified using boron nitride nanosheets aiming to enhancement in the adsorption rate. The prepared hydrogels were investigated by FTIR, XRD, FESEM, TGA/DSC, swelling and cell viability analysis. Cytotoxicity study indicated that prepared sample has a cytocompatibility nature towards healthy normal human cell line (FR2 cells). By changing the pH environment, the drug release properties of the hydrogels can be controlled. The cumulative rate of release for NaCMC-cl-DMAA/AAc hydrogel was 76.5 % at pH = 2.2 and 87.5 % at pH = 7.4. Whereas drug release rate for NaCMC-cl-DMAA/AAc-BNNSs hydrogel composite was 78.6 % at pH = 2.2 and 97.3 % at pH = 7.4 within 4320 min. Gentamicin release kinetics have been determined using the Korsemeyar-Peppas model, which confirms the drug release mechanism.

Staphylococcus aureus Breast Implant Infection Isolates Display Recalcitrance To Antibiotic Pocket Irrigants.

Breast implant-associated infections (BIAIs) are the primary complication following placement of breast prostheses in breast cancer reconstruction. Given the prevalence of breast cancer, reconstructive failure due to infection results in significant patient distress and health care expenditures. Thus, effective BIAI prevention strategies are urgently needed. This study tests the efficacy of one infection prevention strategy: the use of a triple antibiotic pocket irrigant (TAPI) against Staphylococcus aureus, the most common cause of BIAIs. TAPI, which consists of 50,000 U bacitracin, 1 g cefazolin, and 80 mg gentamicin diluted in 500 mL of saline, is used to irrigate the breast implant pocket during surgery. We used in vitro and in vivo assays to test the efficacy of each antibiotic in TAPI, as well as TAPI at the concentration used during surgery. We found that planktonically grown S. aureus BIAI isolates displayed susceptibility to gentamicin, cefazolin, and TAPI. However, TAPI treatment enhanced biofilm formation of BIAI strains. Furthermore, we compared TAPI treatment of a S. aureus reference strain (JE2) to a BIAI isolate (117) in a mouse BIAI model. TAPI significantly reduced infection of JE2 at 1 and 7 days postinfection (dpi). In contrast, BIAI strain 117 displayed high bacterial burdens in tissues and implants, which persisted to 14 dpi despite TAPI treatment. Lastly, we demonstrated that TAPI was effective against Pseudomonas aeruginosa reference (PAO1) and BIAI strains in vitro and in vivo. Together, these data suggest that S. aureus BIAI strains employ unique mechanisms to resist antibiotic prophylaxis treatment and promote chronic infection. IMPORTANCE The incidence of breast implant associated infections (BIAIs) following reconstructive surgery postmastectomy remains high, despite the use of prophylactic antibiotic strategies. Thus, surgeons have begun using additional antibiotic-based prevention strategies, including triple antibiotic pocket irrigants (TAPIs). However, these strategies fail to reduce BIAI rates for these patients. To understand why these therapies fail, we assessed the antimicrobial resistance patterns of Staphylococcus aureus strains, the most common cause of BIAI, to the antibiotics in TAPI (bacitracin, cefazolin, and gentamicin). We found that while clinically relevant BIAI isolates were more susceptible to the individual antibiotics compared to a reference strain, TAPI was effective at killing all the strains in vitro. However, in a mouse model, the BIAI isolates displayed recalcitrance to TAPI, which contrasted with the reference strain, which was susceptible. These data suggest that strains causing BIAI may encode specific recalcitrance mechanisms not present within reference strains.

α-Terpineol synergizes with gentamicin to rescue Caenorhabditis elegans from Pseudomonas aeruginosa infection by attenuating quorum sensing-regulated virulence.

AIMS: This study scrutinized α-Terpineol (α-T) for its anti-virulence and anti-fouling potential against P. aeruginosa PAO1 in conjunction with gentamicin (GeN) using in-vitro, in-silico, and in-vivo approaches. MAIN METHODS: The quorum quenching (QQ) potential of the drug combination was studied using a quorum sensing (QS) biosensor strain and tested for synergy using chequerboard and time-kill kinetics assays. The effect of α-T and GeN on bacterial motility, QS-regulated virulence factor production, and biofilm formation was assessed in P. aeruginosa PAO1 along with molecular docking analysis. The protective effects of α-T-GeN combination were also examined in a Caenorhabditis elegans infection model through slow-killing (SK) assays. KEY FINDINGS: The drug combination displayed synergy, enhanced QQ activity, and suppressed AHL production in PAO1. At sub-inhibitory concentrations, the drug combination suppressed the expression of genes regulating QS and pseudomonal virulence, thereby inhibiting the production of virulence factors in PAO1. The drug combination compromised all forms of pseudomonal motility, strongly inhibited biofilm formation, and successfully eradicated preformed biofilms. Based on these findings, it is concluded that GeN (alone) does not harbor any QQ properties, but enhances the QQ potential of α-T. Moreover, combinational treatment protected C. elegans from pseudomonal infection and improved survival rates by 73 % at 96 h. SIGNIFICANCE: For the first time, the molecular mechanism responsible for the anti-QS activity of α-T was unraveled through a comprehensive investigation, thereby asserting its potential as an anti-virulent drug against P. aeruginosa.

A newly isolated bacteriophage vB8388 and its synergistic effect with aminoglycosides against multi-drug resistant Klebsiella oxytoca strain FK-8388.

The bacteriophage vB8388 can lyse multi-drug resistant Klebsiella oxytoca strain FK-8388 and maintain stability in a wide range of temperatures (from 4 °C to 80 °C) and pHs (3-11). Bioinformatics analysis showed that vB8388 is a linear double-stranded DNA virus that is 39,750 long with 50.65% G + C content and 44 putative open reading frames (ORFs). Phage vB8388 belongs to the family Autographviridae and possesses a non-contractile tail. The latency period of vB8388 was approximately 20 min. The combination of phage vB8388 and gentamicin, amikacin, or tobramycin could effectively inhibit the growth of K. oxytoca strain FK-8388, with a decrease of more than 4 log units within 12 h in vitro. Phage vB8388 showed a strong synergistic effect with gentamicin that could enhance the anti-biofilm effect of vB8388. The phage + gentamicin combination also showed synergy in vivo in the larval infection model of Galleria mellonella. In conclusion, the findings of this study suggest the potential of phage + antibiotic combination therapy to be used as an alternative therapeutic approach for treating infectious diseases caused by multidrug-resistant bacteria.

Investigation of the synergistic effect of glycolipid biosurfactant produced by Shewanella algae with some antibiotics against planktonic and biofilm forms of MRSA and antibiotic resistant Acinetobacter baumannii.

To tackling antibiotic resistance and the appearance of multidrug-resistant (MDR) strains, one current approach is the combined use of biosurfactants with antibiotics to increase their efficacy. The antimicrobial ability of biosurfactant produced by Shewanella algae strain B12 was examined using the agar well diffusion method versus some resistant Gram-negative and Gram-positive bacteria. The Minimum Inhibitory Concentration (MIC) of Glycolipid-Biosurfactant of B12 (GBB12) was performed by the broth dilution technique. The inhibition of biofilm formation, disruption of biofilm, and reducing the population of viable cells in biofilm were evaluated by the microtiter plate method. Finally, Scanning Electron Microscopy (SEM) analysis was used to confirm the disruption of the cell membrane by GBB12. In all experiments, when GBB12 was added to antibiotics (except Amikacin), the antimicrobial activity was increased. The synergistic effects of GBB12 and antibiotics (Ciprofloxacin and Gentamycin) against Methicillin-Resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii were confirmed by the Fractional Inhibitory Concentration Index (FICI). GBB12-Gentamycin mixture almost completely inhibits the formation of A. baumannii biofilm, reaching 99.8% inhibition. Also, the rate of MRSA biofilm inhibition treated with GBB12-Ciprofloxacin mixture was found to be 99.4%. biosurfactant-antibiotic mixture could be adequate replacements for traditional antibiotics in the near future. This study shows the potential of GBB12 as antimicrobial and antibiofilm agent.

Genotypic and phenotypic diversity of Prototheca spp. recovered from bovine mastitis in terms of antimicrobial resistance and biofilm formation ability.

BACKGROUND: The Prototheca algae have recently emerged as an important cause of bovine mastitis globally. Isolates from bovine mastitis in several countries were nearly all identified as P. bovis, suggesting that it was the main causative agent of bovine protothecal mastitis. The aim of the present study was to evaluate the presence and isolation of Prototheca spp. in dairy farms, detect the genetic diversity among strains, determine the capacity of producing biofilm and their resistance to antifungal and antimicrobial drugs. RESULTS: A total of 48 Prototheca isolates from four different farms were randomly selected to be investigated. Multiplex PCR showed all isolated colonies were Prototheca bovis. Performing RAPD-PCR by using OPA-4 primer, it was revealed that there was a clear amplification pattern. Different levels of biofilm production were observed among strains. Among 48 isolates, only 4 of them (8.33%) showed strong biofilm production. By using E-test strips, amphotericin B was able to inhibit the growth of all the strains tested. Disc diffusion method used for antimicrobial sensitivity test showed that the highest activity was demonstrated by gentamicin and colistin with 95.83% (46/48) and 89.58% (43/48) of sensitive strains, respectively. CONCLUSIONS: The present study showed that RAPD-PCR was a rapid tool for discriminating P. bovis strains. Also, gentamicin and colistin can be considered as potential antimicrobial drugs which can prevent the growth of the mentioned strains in vitro, although there is no effective clinical treatment yet. Further studies are needed in order to detect an effective clinical therapy considering biofilm production by Prototheca spp. and their probable role in Prototheca pathogenicity.

Antibacterial Mechanism of Chitosan-Gentamicin and Its Effect on the Intestinal Flora of Litopenaeus vannamei Infected with Vibrio parahaemolyticus.

To explore the application of chitosan-gentamicin conjugate (CS-GT) in inhibiting Vibrio parahaemolyticus (V. parahaemolyticus), which is an important pathogen in aquatic animals worldwide, the antimicrobial activity of CS-GT and the effects of a CS-GT dose on the intestine histopathology and intestinal flora of V. parahaemolyticus-infected shrimps were explored. The results showed that CS-GT possessed broad-spectrum antibacterial activity, with minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and half inhibitory concentration (IC50) of 20.00 ± 0.01, 75.00 ± 0.02 and 18.72 ± 3.17 µg/mL for V. parahaemolyticus, respectively. Further scanning electron microscope and cell membrane damage analyses displayed that the electrostatic interaction of CS-GT with cell membrane strengthened after CS grafted GT, resulting in leakage of nucleic acid and electrolytes of V. parahaemolyticus. On the other hand, histopathology investigation indicated that high (100 mg/kg) and medium (50 mg/kg) doses of CS-GT could alleviate the injury of a shrimp's intestine caused by V. parahaemolyticus. Further 16S rRNA gene sequencing analysis found high and medium dose of CS-GT could effectively inhabit V. parahaemolyticus invasion and reduce intestinal dysfunction. In conclusion, CS-GT possesses good antibacterial activity and could protect shrimps from pathogenic bacteria infection.