Selective Antibiofilm Effects of Lucilia sericata Larvae Secretions/Excretions against Wound Pathogens.

Background. Maggot debridement therapy (MDT), using Lucilia sericata larvae, represents efficient, simple, and low-cost therapy for the treatment of chronic wounds. Aim. The aim was to investigate the antibiofilm activity of maggot excretions/secretions (ES) against biofilm of wound isolates Staphylococcus aureus (S. aureus), Enterobacter cloacae (E. cloacae), and Proteus mirabilis (P. mirabilis). Methods. Quantification of biofilm formation, was carried out using a microtiter plate assay. Proteolytic activity of maggot ES was performed using skim milk agar plates. A solid phase extraction and reverse phase HPLC C18 chromatography were employed to the isolate of maggot ES antibiofilm compounds. Results. Maggot ES at 100 mg/mL concentration significantly reduced biofilm formation thus disrupting established biofilm of E. cloacae. Heat-treated ES did not show any antibiofilm activity towards E. cloacae. Similar results were obtained in the case of S. aureus; however, the heat-treatment of maggot ES did not affect its antibiofilm activity. Moreover, a compound with molecular weight of 25 kDa exhibiting antibiofilm activity was identified in maggot ES. On the other hand, maggot ES protected and even stimulated P. mirabilis biofilm formation. Conclusions. Our results suggest that maggot ES may act selectively against different bacterial strain.

Methylglyoxal may affect hydrogen peroxide accumulation in manuka honey through the inhibition of glucose oxidase.

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 µM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 µM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H2O2 (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

Anti-biofilm effects of honey against wound pathogens Proteus mirabilis and Enterobacter cloacae.

Biofilm growth and its persistence within wounds have recently been suggested as contributing factors to impaired healing. The goal of this study was to investigate the anti-biofilm effects of several honey samples of different botanical origin, including manuka honey against Proteus mirabilis and Enterobacter cloacae wound isolates. Quantification of biofilm formation was carried out using a microtiter plate assay. All honeys at a sub-inhibitory concentration of 10% (w/v) significantly reduced the biofilm development of both isolates. Similarly, at a concentration of 50% (w/v), each of the honeys caused significant partial detachment of Pr. mirabilis biofilm after 24 h. On the other hand, no honey was able to significantly detach Ent. cloacae biofilm. In addition, treatment of Ent. cloacae and Pr. mirabilis biofilms with all honeys resulted in a significant decrease in colony-forming units per well values in a range of 0.35-1.16 and 1.2-7.5 log units, respectively. Of the tested honeys, manuka honey possessed the most potent anti-biofilm properties. Furthermore, methylglyoxal, an antibacterial compound of manuka honey, was shown to be responsible for killing biofilm-embedded wound bacteria. These findings suggest that manuka honey could be used as a potential therapy for the treatment of wounds containing Pr. mirabilis or Ent. cloacae.

Expression of lucifensin in Lucilia sericata medicinal maggots in infected environments.

Lucifensin, a novel larval defensin, is one of the antibacterial agents of medicinal maggots involved in maggot therapy. The goal of this study was to examine lucifensin expression in various larval tissues during Lucilia sericata development and in maggots exposed to a variety of infectious environments in vitro. In situ hybridisation revealed lucifensin expression in the salivary glands of all larval stages. Expression was occasionally detected in a few cells of the fat body and in the grease coupler of the salivary glands. Expression of lucifensin in the salivary glands was initiated 5-6 h after hatching from the egg. Maximum expression was reached about 24 h after hatching, remained strong during the second and third instars and declined at the end of the third instar, before the wandering stage. Expression of lucifensin was also investigated in maggots after oral ingestion of certain pathogens regularly found in infected chronic wounds. No differences were detected in the salivary glands after stimulation by wound bacterial isolates. However, lucifensin expression was strongly stimulated in the fat body by the presence of Staphylococcus aureus and Pseudomonas aeruginosa. Our data suggest that certain infectious environments increase lucifensin expression only in the fat body, whereas its production and antimicrobial activity in excretion/secretion products are not affected.

Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications.

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.

Honeydew honey as a potent antibacterial agent in eradication of multi-drug resistant Stenotrophomonas maltophilia isolates from cancer patients.

Multi-drug resistance in nosocomial pathogens is a continually evolving and alarming problem in health care units. Since ancient times, honey has been used successfully for the treatment of a broad spectrum of infections with no risk of resistance development. This study investigated the antibacterial activity of two natural honeys, namely honeydew and manuka, against 20 nosocomial multi-drug resistant Stenotrophomonas maltophilia (S. maltophilia) isolates from cancer patients. An antibiotic susceptibility test was carried out using the disk diffusion method with 20 antibiotic disks. The antibacterial activity of honey was determined using a broth dilution method. The concentration of honey used in the study was within the range of 3.75% to 25% (w/v). All 20 clinical isolates were multi-drug resistant against 11 to 19 antibiotics. The MICs for honeydew honey ranged from 6.25% to 17.5%, while those for active manuka honey ranged from 7.5% to 22.5%. Honeydew honey had lower MICs than manuka honey against 16 of the tested isolates. This study showed that Slovak honeydew honey has exceptional antibacterial activity against multi-drug resistant S. maltophilia isolates and was more efficient than manuka honey (UMF 15+). Honeydew honey with strong antibacterial activity could be used as a potential agent to eradicate multi-drug resistant clinical isolates.