Vitamin C Enhances the Antibacterial Activity of Honey against Planktonic and Biofilm-Embedded Bacteria.

Multifactorial antibacterial action is an important feature of honey; however, its bactericidal efficacy against biofilm-embedded bacteria is limited. The aim of this study was to investigate the impact of vitamin C (Vit C) on the antibacterial activity of natural honeys against planktonic as well as biofilm-embedded bacterial pathogens. The antibacterial activity of four honey samples supplemented with Vit C was expressed as the minimum inhibitory concentration (MIC). At sub-MICs, Vit C significantly increased the antibacterial activity of the tested honeys against Pseudomonas aeruginosa in planktonic cultures. However, after supplementation, honeydew honey, the most active honey, was ineffective against Staphylococcus aureus. On the other hand, when 100% honeydew honey was supplemented with Vit C (100 mg/g of honey) in a multispecies wound biofilm model, complete eradication of almost all bacterial isolates, including S. aureus, was observed. Furthermore, a mixture of honey and Vit C was partially effective against Enterococcus faecalis, whereas honey alone exhibited no antibacterial activity against this bacterium. Vit C counteracted hydrogen peroxide in honey solution and, thus, eliminated the major antibacterial compound present in honey. It is likely that a combination of honey with Vit C may trigger the intracellular production of reactive oxygen species in bacterial cells, but the exact cellular mechanisms warrant further investigations.

Antibacterial potential of Swiss honeys and characterisation of their bee-derived bioactive compounds.

BACKGROUND: Antibacterial activity of honey is not only crucial characteristic in selection of honey for medical usage but also an important honey quality marker. The aim of the study was to characterise the antibacterial potential of 29 honey samples representing the main types of multi-floral blossom and honeydew honeys produced in Switzerland. Antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa was expressed as a minimum inhibitory and bactericidal concentrations (MIC and MBC). Furthermore, the content of bee-derived glucose oxidase (GOX) and its enzymatic product, H2 O2 , were also evaluated. RESULTS: All honey samples successfully met basic defined criteria (moisture and hydroxymethylfurfural (HMF)) tested in this study. Honeydew honeys were the most effective honey samples and generated the highest levels of H2 O2 . A strong significant correlation was found between the overall antibacterial activity and the level of H2 O2 among all honey samples. Interestingly, the content of GOX in honey samples did not correlate with their antibacterial activity as well as H2 O2 production capacity. A weak antibacterial activity was determined in five floral honeys, most likely due to increased enzymatic activity of pollen-derived catalase. CONCLUSION: This study showed that antibacterial effect of Swiss honey samples is associated mainly with H2 O2 . © 2019 Society of Chemical Industry.

Antibacterial Activity of Different Blossom Honeys: New Findings.

Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the content of GOX and level of H2O2. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H2O2 levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

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.

Dimethyl sulfoxide attenuates TNF-α-induced production of MMP-9 in human keratinocytes.

Dimethyl sulfoxide (DMSO), an aprotic solvent, is found to be useful as a topical agent with antioxidant effects in treatment of chronic wounds. However, the effects of DMSO on matrix metalloproteinase-9 (MMP-9) production in the presence of an inflammatory environment as in the case of disordered wound healing has not been previously investigated. The aim of this study was to investigate whether TNF-α-induced MMP-9 levels and MMP-9 mRNA expression from human keratinocytes (HaCaT) might be attenuated by DMSO. Human keratinocytes were treated with DMSO (0.1-1%) for 24 h and then exposed to tumor necrosis factor (TNF)-α (10 ng/ml) for an additional 24 h. Expression and production of MMP-9 from HaCaT cells were determined by reverse transcription polymerase chain reaction (RT-PCR) and gelatin zymography, respectively. Results showed that DMSO inhibited production of both MMP-9 levels and MMP-9 mRNA expression in TNF-α-stimulated cells in a concentration-dependent manner. Inhibition of MMP-9 levels was statistically significant at DMSO concentrations of 0.75% and higher. Similarly, the increase of MMP-9 mRNA expression levels in TNF-α-stimulated cells was markedly reduced by DMSO. Data suggest that DMSO may attenuate the deleterious effects of MMP-9 through downregulation at the transcription level. Therefore, DMSO may provide a good strategy to prevent TNF-α-induced proteolytic activity in cutaneous inflammatory reactions.

Phenotypic and molecular characterization of human salmonella enterica serovar 4,[5],12:i:- isolates in Slovakia.

Forty-three epidemiologically unrelated emerging Salmonella enterica subsp. enterica serovar 4,[5],12:i:- strains isolated during the period 2009-2010 in Slovakia were characterized by phenotypic and genotypic methods. Thirty-one isolates (72.1%) expressed resistance to ampicillin, streptomycin, sulfizoxazole, and tetracycline [R-type ASSuT]. The majority of the strains belonged to both definitive phage types DT193 (30.2%) and U311 (27.9%). Other phage types identified were U302 (6.9%), DT18 (4.7%), and DT194 (2.3%). Twelve strains (27.9%) were not typeable. Pulsed-field gel electrophoresis analysis identified three closely related major banding profiles (X1, X1a, and X2), suggesting the similarity and close epidemiological relationship between S. enterica serovar 4,[5],12:i:- strains. In two isolates with R-type ASSuT, phage type NT and in one isolate with R-type ACROSSuSxTTTMPNA, phage type DT193 class 1 integrons were found encoding bla(PSE-1) and dfrA, aadA1, respectively.

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.

Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey.

Honeydew honey is increasingly valued due to its pronounced antibacterial potential; however, the underlying mechanism and compounds responsible for the strong antibacterial activity of honeydew honey are still unknown. The aim of this study was to investigate the inhibition of bacterial growth of 23 honeydew honey samples. Activity of bee-derived glucose oxidase (GOX) enzyme, the content of defensin-1 (Def-1) and hydrogen peroxide (H2O2), and total polyphenol content were determined in the 23 honey samples. Our results demonstrated that antibacterial activity of honeydew honey was equivalent to medical-grade manuka and kanuka honey and was abolished by catalase. Although H2O2 is an important factor in the inhibition of bacterial growth, polyphenolic compounds and their interaction with H2O2 are the key factors responsible for high antibacterial activity of honeydew honey. In addition, our results indicated that the antibacterial activity of honeydew honey is not dependent on GOX-mediated production of H2O2 or the presence of Def-1.

Variability in occurrence of multiple prophage genes in Salmonella Typhimurium strains isolated in Slovak Republic.

Lysogenic bacteriophages are a significant source of variability in closely related Salmonella strains. In this study, screening for diversity of 152 Salmonella Typhimurium strains was performed using PCR detection of selected prophage regions derived from phages P22, Gifsy-1, Gifsy-2, Fels-1, ST104 and SopEPhi. A high degree of variability was observed in the presence of specific genes. Based on the presence of particular prophage genes, we divided strains into 37 different PCR-prophage profiles; 20 of them were represented by only a single strain. Using multilocus variable number tandem repeats analysis (MLVA), 152 Salmonella strains were separated into 82 MLVA strings. Similar grouping of Salmonella strains was observed in the case of PCR-prophage detection and MLVA and the results corresponded well with the phage type of strains. However, several Salmonella strains were detected, which were closely related according to MLVA; yet, they differed in PCR phage profiles. The observations support a view that integration/excision of bacteriophages in Salmonella strains are frequent events shaping the bacterial genome.

Molecular characterization of class 1 integrons in clinical strains of Salmonella typhimurium isolated in Slovakia.

The presence of class 1 integrons was investigated in 156 epidemiologically unrelated Salmonella Typhimurium isolates. Of these 156 isolates, 70 were of definitive phage type DT104 and 86 were strains of various phage type, RDNC and untypable, designated here as non-DT104 strains. Integrons were found in 47 of DT104 isolates (67.1%), while in all strains with characteristic pentaresistance (R-type ACSSuT) two integrons 1.0 kb and 1.2 kb in size were found. Among 86 non-DT104 strains, integrons with sizes of 1.6 kb and 1.9 kb in four multidrug-resistant strains DT193 and U302 were found. The integrons from selected strains were further sequenced and the aadA1, aadA2, dhfr1, dhfr12 and bla(PSE) genes were found embedded in cassettes.

Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo.

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closure in vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion.

Salmonella enterica serovar Kentucky: antimicrobial resistance and molecular analysis of clinical isolates from the Slovak Republic.

A collection of 68 isolates of Salmonella enterica serovar Kentucky collected during the period 2003-2004 from humans in two geographical regions in the Slovak Republic was studied. The original isolate of this serovar was associated with travel to Egypt, and the emergence of other isolates was due to the nosocomial spread of this strain in two hospitals. Antimicrobial susceptibility testing, class I integrons content, pulsed-field gel electrophoresis (PFGE) analysis and plasmid DNA profiles were performed on all isolates. A high proportion (89.7%) of the isolates was multidrug-resistant, while 67 strains expressed resistance against ciprofloxacin. By sequence analysis of randomly selected strains, the point mutations in quinolone resistance-determining region of the DNA gyrase were found. The S. Kentucky isolates investigated were determined to be clonally related by PFGE as well as plasmid DNA analysis.

Analysis of integrons in human isolates of Salmonella enterica serovar typhimurium isolated in the Slovak Republic.

About 110 sporadic, epidemiologically unrelated Salmonella enterica serovar typhimurium strains isolated in the Slovak Republic were analyzed for the presence of integrons. Of these 110 examined strains, 47 were of definitive phage type DT104 and 63 were strains of various phage type, RDNC and untypeable, designated here as non-DT104 strains. All isolates were also tested for antimicrobial resistance to 10 antibiotics as well as for the presence of virulence plasmid. Of 63 non-DT104 strains, 15 isolates were multiple-resistant, independently from phage type, other strains were resistant to one, two or three drugs. Resistance to ampicillin, streptomycin, tetracycline and sulfisoxazole was most frequently observed. Among the DT104 isolates up 65.9% exhibited characteristic pentaresistance--ACSSuT phenotype. The integron content was studied in PCR experiments using a 5'-CS/3'-CS primer pair. Fourteen non-DT104 strains, independently from phage type, were found to carry integrons with amplicons 650-1900 bp in size. Thirty-six DT104 strains contained integrons of 1000 and 1200 bp and 31 of they exhibited the ACSSuT phenotype. No integron was found in 10 DT104 strains, which included strains mostly resistant only to streptomycin, tetracycline and sulfisoxazole. The majority of non-DT104 strains did not possess any integrons. Our findings show the widespread existence of both resistant and multiple-resistant epidemiologically unrelated Salmonella typhimurium strains and suggest that integrons contribute to this antimicrobial resistance. The presence of 90-kb virulence plasmid in the 54 non-DT104 and in the all DT104 strains was found.

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.

Antimicrobial resistance of human Salmonella enterica serovar Typhimurium U302 strains: prevalence of R-type ASSuT in Slovakia, 2006-2011.

Salmonella enterica serovar Typhimurium is a common cause of non-typhoid salmonellosis in humans. Since 2006, an increase in the human infections caused by U302 phage type in Slovakia has been documented and, from 2006 to 2011, a total of 291 U302 human strains were isolated. Antimicrobial susceptibility testing demonstrated that these strains had a high overall antimicrobial resistance and 244 (83.8%) of the tested strains were multidrug-resistant (MDR). The most prevalent resistance was to ampicillin, streptomycin, sulfisoxazole, and tetracycline (R-type ASSuT), which was verified in 87 (29.9%) strains. The annual rate of this resistance type varies, but the largest number of these strains were identified in 2009 and 2010. The classical pentaresistance phenotype (R-type ACSSuT), characteristic of the DT104 phage type, was found only in 40 (13.7%) U302 strains. These results suggested that although the prevalence of DT104 phage type has decreased, ASSuT as well as ACSSuT resistance markers continue to circulate. Therefore, continual surveillance of the occurrence of these and similar MDR phage types is necessary.

Fir honeydew honey flavonoids inhibit TNF-α-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing.

Matrix metalloproteinase-9 (MMP-9) appears to be a major protease responsible for the degradation of matrix and growth-promoting agents in chronic wounds. Honey has been successfully used for treating non-healing wounds associated with infections. However, the mechanisms of its action at the cellular level have remained poorly understood. The aim of this study was to investigate the effect of fir honeydew honey on TNF-α-induced MMP-9 expression and secretion from human keratinocytes (HaCaT) and to identify the honey component(s) responsible for a discovered effect. A C18 solid-phase column was used for preparation of honey aqueous extract (HAE). Expression and production of MMP-9 by HaCaT cells were determined by reverse transcription-PCR, gelatine zymography and Western blot analysis using a polyclonal antibody against MMP-9. We found that HAE inhibited TNF-α-induced production of MMP-9 in keratinocytes in a dose-dependent manner at both the mRNA and protein levels. Apigenin and kaempferol, identified flavonoids in HAE, markedly inhibited MMP-9 production from HaCaT and epidermal keratinocytes. Taken together, fir honeydew honey, which contains certain flavonoids, prevents TNF-α-induced proteolytic activity in cutaneous inflammation. Thus, our findings provide clear evidence that honey may serve as a natural treatment for dermatological problems associated with a persistent inflammation.

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.

Trends in phage types of Salmonella enterica serovars Enteritidis and Typhimurium isolated in Slovakia from 1995 to 2009.

The phage typing of 3900 isolates of Salmonella Enteritidis and 1741 isolates of Salmonella Typhimurium has been carried out in the period 1995-2009. Among Salmonella Enteritidis in individual years, the most prevalent phage type (PT) was 8. The most predominant PTs of Salmonella Typhimurium were DT104 and U302.

Increasing trend of resistance to nalidixic acid and emerging ceftriaxone and ciprofloxacin resistance in Salmonella enterica serovar Typhimurium in Slovakia, 2005 to 2009.

A 5-year survey from 2005 to 2009 revealed an increasing trend of resistance to nalidixic acid (from 0% in 2005 to 11% in 2009) among 858 clinical isolates of Salmonella Typhimurium. In addition, 10 ceftriaxone-resistant and 3 ciprofloxacin-resistant Salmonella Typhimurium isolates were detected over the period of study.

Detection of the class 1 integrons and SGI1 among Salmonella enterica Serovar Typhimurium DT104, U302, DT120, DT193, and nontypable human isolates.

Salmonella enterica serovar Typhimurium strains of particular phage types, such as DT104, U302, DT120, DT193, and nontypable strains, are often characterized by resistance to multiple antibiotics. This antibiotic resistance can be caused by the presence of the integrons, transposons, Salmonella genomic island 1 (SGI1), or conjugative plasmids. In this study we were interested in the relative contribution of integrons and SGI1 to the antibiotic resistance of the four mentioned phage types and nontypable S. Typhimurium human strains. Altogether 193 isolates were characterized for antibiotic susceptibility, presence of class 1 integrons, and the left junction of SGI1. Based on the presence of class 1 integrons and the left junction of SGI1, all strains could be clustered into three groups. The first group consisted of 69 strains positive for both the class 1 integrons and the left junction of SGI1. The strains of this group belonged mainly to DT104, U302, and DT120 phage types with resistance phenotype ACSSuT or ACSSuTNA. The second group comprised 9 strains which were positive only for the presence of class 1 integrons. In this group were some strains of multiple-antibiotic-resistant phage types: DT120, DT193, U302; and nontypable. The third group consisted of 115 strains in which neither the class 1 integrons nor the left junction of SGI1 were detected. Although the isolates were resistant to 2-8 antibiotics, the most frequent resistance type of these strains were ASSuT and SSu. By nucleotide sequencing of class 1 integrons PCR amplicons, the following embedded gene cassettes were determined: aadA1, aadA2, aadA5, aadA7, bla(PSE-1), sat1, dfrA1; and dfrA14. Our study shows a high prevalence and diversity of class 1 integrons embedded antimicrobial gene cassettes and their strong association with SGI1.