Antistaphylococcal Activity Research Articles

Design, synthesis and structure–activity relationships of substituted oxazole–benzamide antibacterial inhibitors of FtsZ

The design, synthesis and structure–activity relationships of a series of oxazole–benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.

Biological activity of antibacterial peptides matches synergism between electrostatic and non electrostatic forces

Substitution of Ala 108 and Ala 111 in the 107–115 human lysozyme (hLz) fragment results in a 20-fold increased anti-staphylococcal activity while its hemolytic activity becomes significant (30%) at very high concentrations. This analog displays an additional positive charge near the N-terminus (108) and an extra Trp residue at the center of the molecule (111), indicating that this particular amino acid sequence improves its interaction with the bacterial plasma membrane. In order to understand the role of this arrangement in the membrane interaction, studies with model lipid membranes were carried out.The interactions of peptides, 107–115 hLz and the novel analog ([K108W111]107–115 hLz) with liposomes and lipid monolayers were evaluated by monitoring the changes in the fluorescence of the Trp residues and the variation of the monolayers surface pressure, respectively. Results obtained with both techniques revealed a significant affinity increase of [K108W111]107–115 hLz for lipids, especially when the membranes containing negatively charged lipids, such as phosphatidylglycerol. However, there is also a significant interaction with zwitterionic lipids, suggesting that other forces in addition to electrostatic interactions are involved in the binding. The analysis of adsorption isotherms and the insertion kinetics suggest that relaxation processes of the membrane structure are involved in the insertion process of novel peptide [K108W111]107–115 hLz but not in 107–115 hLz, probably by imposing a reorganization of water at the interphases.In this regard, the enhanced activity of peptide [K108W111]107–115 hLz may be explained by a synergistic effect between the increased electrostatic forces as well as the increased hydrophobic interactions.

Pharmacokinetics and in vivo antistaphylococcal efficacy of TXY541, a 1-methylpiperidine-4-carboxamide prodrug of PC190723

The benzamide derivative PC190723 was among the first of a promising new class of FtsZ-directed antibacterial agents to be identified that exhibit potent antistaphylococcal activity. However, the compound is associated with poor drug-like properties. As part of an ongoing effort to develop FtsZ-targeting antibacterial agents with increased potential for clinical utility, we describe herein the pharmacodynamics, pharmacokinetics, in vivo antistaphylococcal efficacy, and mammalian cytotoxicity of TXY541, a novel 1-methylpiperidine-4-carboxamide prodrug of PC190723. TXY541 was found to be 143-times more soluble than PC190723 in an aqueous acidic vehicle (10mM citrate, pH 2.6) suitable for both oral and intravenous in vivo administration. In staphylococcal growth media, TXY541 converts to PC190723 with a half-life of approximately 8h. In 100% mouse serum, the TXY541-to-PC190723 conversion was much more rapid (with a half-life of approximately 3min), suggesting that the conversion of the prodrug in serum is predominantly enzyme-catalyzed. Pharmacokinetic analysis of both orally and intravenously administered TXY541 in mice yielded a half-life for the PC190723 conversion product of 0.56h and an oral bioavailability of 29.6%. Whether administered orally or intravenously, TXY541 was found to be efficacious in vivo in mouse models of systemic infection with both methicillin-sensitive and methicillin-resistant S. aureus. Toxicological assessment of TXY541 against mammalian cells revealed minimal detectable cytotoxicity. The results presented here highlight TXY541 as a potential therapeutic agent that warrants further pre-clinical development.

Impact of Hyperbaric Oxygenation on Blood Antistaphylococcal Activity During Liver Resection

Objective: to study the impact of liver resection (LR) and a hyperbaric oxygenation (HBO) session and their combination on blood antistaphylococcal activity. Material and methods. Experiments were carried out on 77 outbred albino rats (females) exposed to LR (15—20% of the liver weight) and HBO at 3 ata as a 50-min session once daily, which was used in the operated rats in the first 3 days after LR. Serum bactericidal activity against opportunistic (No. 1726) and pathogenic (No. 209) S.aureus strains and the ability of neutrophils and monocytes to absorb S.aureus 209 were studied. Results. restoring the serum bactericidal activity against S.aureus 1726, HBO used after LR enhances its activity against pathogenic S.aureus 209. By decreasing the inhibitory effect of LR on the ability of the liver to enrich blood with the neutrophils that actively phagocy-tize the staphylococcus, HBO eliminates the retention of the monocytes actively phagocytizing S.aureus 209 in the operated organ, by concurrently increasing the count of these cells in the arterial blood. HBO stimulates the rate of uptake of S.aureus 209 by neutrophils in the operated organism when the blood passes through the vessels of the portal system with the partial retention of these cells in the liver portion left after its resection. By selectively suppressing the humoral component of blood antistaphylococcal defense, HBO in healthy unoperated animals stimulates the ability of the liver to enrich the blood with the monocytes that actively phagocytize the staphylococcus. At the same time the rate of uptake of S.aureus 209 by neutrophils is stimulated by hyperoxia. Conclusion. HBO regulates blood antistaphylococcal activity changes in response to LR by exerting a selective effect on the bactericidal activity of the blood against the staphylococcus in rats. Key words: liver, resection, hyperoxia, bactericidal activity, blood, staphylococcus.

INVESTIGATIONS INTO THE ANTIMICROBIAL ACTIVITY OF METHANOL LEAF EXTRACT OF CALLISTEMON RIGIDUS

Traditional medicine is widely used in under developed countries and in developing and developed countries as alternative or complementary medicine. A lot of the herbs used have proven pharmacological activity. The bat tle against microbes is one that man cannot afford to lose, hence the continual search for newer antibiotics. Callistemon rigidus has been discovered to have a very good antimicrobial activity. The activity of methanol leaf extract of Callistemon rigidus a gainst representative Gram - nega tive and Gram - positive bacteria and Fungus was determined. Gram - negative organism; E. coli and Pseudomonas aeruginosa were more sensitive to the leaf extract. Candida albicans was least sensitive. This study supports the use of Callistemon rigidus traditionally and verifies that the methanol leaf extract has antimicrobial, including antistaphylococcal activity.

An FtsZ-Targeting Prodrug with Oral Antistaphylococcal Efficacy In Vivo

The bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamide PC190723 was among the first FtsZ-targeting compounds to exhibit in vivo efficacy in a murine infection model system. Despite its initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We describe here the development of an N-Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivo efficacy. The N-Mannich base derivative (TXY436) is ∼100-fold more soluble than PC190723 in an acidic aqueous vehicle (10 mM citrate, pH 2.6) suitable for oral in vivo administration. At physiological pH (7.4), TXY436 acts as a prodrug, converting to PC190723 with a conversion half-life of 18.2 ± 1.6 min. Pharmacokinetic analysis following intravenous administration of TXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respectively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Using a mouse model of systemic infection with methicillin-sensitive Staphylococcus aureus or methicillin-resistant S. aureus, we show that TXY436 is efficacious in vivo upon oral administration. In contrast, the oral administration of PC190723 was not efficacious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentrations at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 a worthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections.

AFN-1252 in vitro absorption studies and pharmacokinetics following microdosing in healthy subjects

ObjectivesTo investigate the absorption, distribution, metabolism and excretion of AFN-1252, a novel inhibitor of the essential FabI enzyme in Staphylococcus spp., in vitro and following microdosing in healthy adult male subjects following intravenous and oral administration. MethodsThree ADME studies, comprising a Caco-2 assay, a rat intestinal perfusion model and a microdosing study in healthy human volunteers, were conducted. ResultsThe Caco-2 assay indicated that AFN-1252 in solution is well-absorbed and undergoes insignificant efflux, and its transport across the intestinal wall is probably passive. In the rat intestinal perfusion model, AFN-1252 exhibited high permeability potential across three segments, in the rank order of jejunum=ileum>colon. Taken together with the low aqueous solubility, the data from these studies indicate that AFN-1252 is a BCS Class II molecule with solubility-limited absorption.Analysis of the [14C]-AFN-1252 radioactivity concentration–time data indicated similar pharmacokinetics following intravenous and oral administration in the microdosing study in healthy volunteers. These included long terminal half-lives of ∼7h and 83% bioavailability, indicating that there was little first-pass metabolism following oral dosing. AFN-1252 exhibited good distribution to skin and skin structures where its anti-staphylococcal activity may be required. Urinary and faecal excretion are major elimination routes for [14C]-AFN-1252 following intravenous or oral administration. ConclusionsAFN-1252 has the potential for both intravenous and oral administration, once- or twice-daily dosing and good tissue distribution in humans. Further safety, efficacy and pharmacokinetic studies in man are required to investigate therapeutically-relevant doses for this novel agent and its targeted selectivity and high potency against Staphylococcus spp.

Synthesis of Small Combinatorial Libraries of Natural Products: Identification and Quantification of New Long‐chain 3‐Methyl‐2‐alkanones from the Root Essential Oil of Inula helenium L. (Asteraceae)

Recently, a potent anti-staphylococcal activity of Inula helenium L. (Asteraceae) root essential oil was reported. Also, bioassay guided fractionation of the oil pointed to eudesmane sesquiterpene lactones and a series of unidentified constituents as the main carriers of the observed activity. To identify nine new constituents (long-chain 3-methyl-2-alkanones) from a fraction of this root essential oil with a low minimum inhibitory concentration value (0.8 µg/mL) by employing a synthetic methodology that leads to the formation of a small combinatorial library of these compounds. The identity of these constituents was inferred from mass spectral fragmentation patterns and GC retention data. A library of 3-methyl-2-alkanones (C11 -C19 homologous series) was synthesised in three steps starting from methyl acetoacetate and the corresponding alkyl halides. The synthetic library was also screened for in vitro anti-microbial activity. Gas chromatographic analyses of I. helenium essential oil samples with spiked compounds from the synthesised library corroborated the tentative identifications of the long-chain 3-methyl-2-alkanones. The availability of these anti-microbial compounds from this library made it possible to construct GC/FID calibration curves and determine their content in the plant material: 0.08 - 24.2 mg/100 g of dry roots. The small combinatorial library approach enabled the first unequivocal identification of long-chain 3-methyl-2-alkanones as plant secondary metabolites, and, also, allowed determination of not only a single compound and biological properties, but those of a group of structurally related compounds.

New silica nanostructure for the improved delivery of topical antibiotics used in the treatment of staphylococcal cutaneous infections

In this paper, we report the synthesis, characterization (FT-IR, XRD, BET, HR-TEM) and bioevaluation of a novel γ-aminobutiric acid/silica (noted GABA-SiO₂ or γ-SiO₂) hybrid nanostructure, for the improved release of topical antibiotics, used in the treatment of Staphylococcus aureus infections. GABA-SiO₂ showed IR bands which were assigned to Si-O-Si (stretch mode). The XRD pattern showed a broad peak in the range of 18-30° (2θ), indicating an amorphous structure. Based on the BET analysis, estimations about surface area (438.14 m²/g) and pore diameters (4.76 nm) were done. TEM observation reveals that the prepared structure presented homogeneity and an average size of particles not exceeding 10nm. The prepared nanostructure has significantly improved the anti-staphylococcal activity of bacitracin and kanamycin sulfate, as demonstrated by the drastic decrease of the minimal inhibitory concentration of the respective antibiotics loaded in the GABA-SiO₂ nanostructure. These results, correlated with the high biocompatibility of this porous structure, are highlighting the possibility of using this carrier for the local delivery of the antimicrobial substances in lower active doses, thus reducing their cytotoxicity and side-effects.

Evolution of a fatal septic arthritis caused by a Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus strain

We report the observation of a septic arthritis of the knee complicated within first 36hours by multiorgan failure including acute respiratory distress syndrome (ARDS), heart failure, acute renal failure and disseminated intravascular coagulation (DIC). A diagnosis of staphylococcal arthritis was suspected confirmed by direct examination, and culture showed a Staphylococcus aureus sensitive to methicillin. The sample sent to the National Reference Centre for Staphylococci (Lyon, France) for genetic analysis confirmed the isolate positive for the PVL gene expression. The fulminating evolution of a septic S. aureus arthritis in an otherwise healthy man should probably evoke the possibility of LPV strain. Anti-PLV antibiotics with anti-staphylococcal activity, such as clindamycin and linezolid should be started without waiting for typing of the S. aureus strain.

Identification of 1-chloro-2-formyl indenes and tetralenes as novel antistaphylococcal agents exhibiting sortase A inhibition

Tetralene and indene compounds have shown inhibitory activity against human pathogen, Mycobacterium tuberculosis. Their potential use as antistaphylococcal agent against drug-resistant Staphylococcus aureus has not been explored so far. We determined in vitro antistaphylococcal activity and mechanism of action of these compounds as sortase A inhibitors through in silico analysis followed by biological assays. Tetralene and indene series were tested against S. aureus strains MTCC96, MRSA, and VA30. Three compounds showed significant reduction in MIC in both wild-type and drug-resistant S. aureus strains. In silico absorption, distribution, metabolism, excretion, and toxicity analysis of identified leads and cytotoxicity testing with colorimetric method using Vero and WRL-68 cell lines showed no significant cytotoxic effects. Molecular docking of these molecules with sortase A (PDB: 2KID) showed H-bond interaction with functional site residue Arg197 of sortase A. Sortase A inhibition assay was developed by expressing SrtA∆N from S. aureus strain MTCC96. Tetralene and indene compounds were found to have sortase A inhibitory potential. S. aureus strain MTCC96 treated with these compounds showed surface-sorting inhibition of fibronectin-binding protein and reduction in adherence to host extracellular matrix protein, fibronectin. 1-Chloro, 2-formyl, 6-methoxy, 1-tetralene (Tet-5), 1,5-dichloro, 2-formyl, 1-indene (Tet-20) and 1-chloro, 2-formyl, 5,6-methylenedioxy, and 1-indene (Tet-21) exhibited antistaphylococcal activity along with sortase A inhibition. The results also indicate the possible role of these leads in other reactions essential for cell viability.

Enterococcal and streptococcal resistance to PC190723 and related compounds: Molecular insights from a FtsZ mutational analysis

New antibiotics with novel mechanisms of action are urgently needed to overcome the growing bacterial resistance problem faced by clinicians today. PC190723 and related compounds represent a promising new class of antibacterial compounds that target the essential bacterial cell division protein FtsZ. While this family of compounds exhibits potent antistaphylococcal activity, they have poor activity against enterococci and streptococci. The studies described herein are aimed at investigating the molecular basis of the enterococcal and streptococcal resistance to this family of compounds. We show that the poor activity of the compounds against enterococci and streptococci correlates with a correspondingly weak impact of the compounds on the self-polymerization of the FtsZ proteins from those bacteria. In addition, computational and mutational studies identify two key FtsZ residues (E34 and R308) as being important determinants of enterococcal and streptococcal resistance to the PC190723-type class of compounds.

Actividad antiestafilocóccica y antibiopelícula de los extractos de Juglans netropica DIELS, Piper lineatum RUIZ&PAV. Y Terminalia catappa L.

En este estudio, se investigó la actividad antiestafilocóccica in-vitro de los extractos etanólicos e hidroalcohólicos de 3 plantas medicinales peruanas: Juglans neotropica Diels (corteza), Piper lineatum Ruiz & Pav. (hojas) y Terminalia catappa L. (hojas); recolectadas en las regiones de Amazonas y Cajamarca, en el Perú. La actividad antiestafilocóccica se evaluó mediante el método de microdilución. Los microorganismos utilizados fueron las bacterias Staphylococcus aureus ATCC 25923 y Staphylococcus epidermidis ATCC 12228. Los extractos investigados presentaron actividad significativa frente a ambas bacterias, con una Concentración Mínima Inhibitoria (CMI) de 125 a 500 μg/mL para Staphylococcus aureus, teniendo mayor actividad el extracto etanólico de Juglans neotropica Diels, y de 250 a 500 μg/mL para Staphylococcus epidermidis, teniendo mayor actividad el extracto hidroalcohólico de Piper lineatum Ruiz & Pav. El análisis fitoquímico confirmó la presencia de compuestos terpénicos, flavonoides y alcaloides en los 6 extractos estudiados. La determinación de la Concentración Mínima Inhibitoria de Biopelícula (CMIB) se efectuó por el método de microdilución en placa modificado, utilizando cepas clínicas de Staphylococcus aureus y Staphylococcus epidermidis productoras de biopelícula. Solamente los extractos etanólicos e hidroalcohólicos de las hojas de Piper lineatum L. poseen actividad significativa con CMIB de 500 μg/mL para ambas cepas. Los extractos etanólicos e hidroalcohólicos de Piper lineatum L. pueden ser buenos candidatos para la búsqueda de metabolitos que sirvan para combatir infecciones asociadas a biopelículas de Staphylococcus aureus y Staphylococcus epidermidis.

In vitro antistaphylococcal effects of a novel 45S5 bioglass/agar- gelatin biocomposite films

To assess the antibacterial efficacy of new composite materials developed from microparticles of 45S5 bioactive glass (BG) and agar-gelatin films. In vitro antibacterial activity was evaluated against Staphylococcus spp. because of the importance of this pathogen in damaged tissues and in failures associated with biomaterial implants. To our knowledge, this is the first paper reporting on the suitable combination of BG and agar-gelatin for bioactive and antibacterial films. Bacterial suspensions up or below 10(5) CFU ml(-1) reflecting situations of wound infection and of noninfection, respectively, were prepared and then put in contact with the biomaterials at 37°C. After 24 and 48 h of incubation, the pH value was measured and the staphylococci strains viability was determined by counting in Mueller-Hinton agar plates. Moreover, the biomaterials were prepared for observation under scanning electron microscopy (SEM). Biocomposites (BCs) showed a strong antibacterial effect against all staphylococci strains tested. Some differences were found depending on the strain, the inoculum size and the contact time. This effect was correlated with an alkalinization of the media. By SEM analyses, no bacterial presence was observed on the surface of BCs in any of the cell concentrations tested at any time. Overall, the coating of 45S5 BG on agar-gelatin films promoted BCs with strong antistaphylococcal activity. The effect was efficient under bacterial concentration up or below 10(5) CFU ml(-1). Additionally, none of the strains were found on BCs surfaces. 45S5 bioglass/agar-gelatin biocomposite films are reported for the first time. The results suggest a potential application as wound dressing.

In Vitro Anti-Staphylococcal Activity of Alkaloids from the Leaves of Callestimone Rigidus R.Br

Multidrug resistant Staphylococcus aureus poses a severe global threat worldwide due to their prevalence, genomic plasticity and limited therapeutic options being refractory to most antibiotic classes. This necessitates the discovery of new anti-staphylococcal interventions. Callistemon rigidus R.Br. (Myrtaceae) has been found to possess antibacterial potential against clinical isolates. Thied me study was a isolate and evaluate the antibacterial alkaloids from the leaves of Callistemon rigidus and assess their in vitro anti-staphylococcal potential. Alkaloid isolation was carried out by modiied method for plant alkaloid extraction. The in vitro anti-staphylococcal potential of the alkaloid bioactive fraction was assessed by using micro broth dilution and plate count assay methods. Pus and wound isolates had MIC of 80 µg/mL and 50 IC of 27.22 µg/mL respectively. Burn isolates showed MIC of 320 µg/mL and 50 50 13.57 µg/mL respectively. Urine and vaginal isolates exhibited a MIC of 80 µg/mL 50 and IC of 13.15 µg/mL respectively. Ceixime had MIC values was 320 µg/mL, 160 50 50 µg/mL and 160 µg/mL for pus and wound, burn, urine and vaginal isolates indicating the refractory behaviour when compared to alkaloid bioactive fraction from the leaves of Callistemon. The alkaloid bioactive fraction exhibits immense activity compared to standard antibiotic Ceixime. This work is the irst report of alkaloids and their antimicrobial activity from Callistemon rigidus leaves. The results suggest further isolation of individual alkaloids from alkaloid bioactive fraction and assessment their anti-staphylococcal activity as leads for development of anti-staphylococcal drugs.

Bioinformatic evaluation of the secondary metabolism of antistaphylococcal environmental bacterial isolates

The increasing occurrence of drug-resistant Staphylococcus aureus is exacerbated with a declining rate of antibiotic discovery, particularly those with new mechanisms of action. The decline in antibiotic discovery from traditional sources, such as soil actinobacteria, necessitates examination of lesser studied microbes. Here, we present a strategy to select for organisms that may have a propensity to result in new antistaphylococcal agents by using S. aureus as a bait organism, and selecting organisms that have a natural lytic activity towards it. We have isolated over 80 environmental isolates and typed these organisms using 16S rDNA sequence comparison and deployed bioinformatics to assess the secondary metabolic potential of the isolated antistaphylococcal bacteria using genomic sequences. Bioinformatic analysis highlights the enriched and unique suite of potential antibiotic polyketides and nonribosomal peptides and lantibiotic gene clusters from these organisms. Profiling organic microbial extracts further showed that many of the organisms from the 10 staphylolytic genera secrete agents with antistaphylococcal activity and may serve as new sources for future antistaphylococcal drug discovery.

Design, synthesis and antistaphylococcal activity of marine pyrrole alkaloid derivatives

A novel set of 16 hybrids of bromopyrrole alkaloids with aroyl hydrazone were designed, synthesized and evaluated for antibacterial and antibiofilm activities against methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43866), methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 35556) and Staphylococcus epidermidis (SE, S. epidermidis ATCC 35984). Of the 16 tested hybrids, 14 exhibited equal or superior antibiofilm activity against MSSA and MRSA relative to standard vancomycin. Compound 4m showed highest potency with antibiofilm activity of 0.39 µg/mL and 0.78 µg/mL against MSSA and MRSA, respectively. Thus, this compound could act as a potential lead for further development of new antistaphylococcal drugs.

Comparison of sertaconazole 2% cream versus hydrocortisone 1% ointment in the treatment of atopic dermatitis

Introduction: Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Topical corticosteroids as the mainstay of treatment have many side effects, both cutaneous and systemic. Sertaconazole is an antifungal cream with anti-inflammatory, antipruritic and antistaphylococcal activity. In this study, the authors compare the effect of hydrocortisone ointment and sertaconazole cream in the treatment of AD. Materials and methods: This study was a double-blind bilateral comparison study. Forty-five patients applied sertaconazole 2% cream twice daily on one side of the body and hydrocortisone 1% ointment twice daily on the opposite side for 1 month. The authors used a modified SCORAD score to assess the severity of the disease before and after therapy. Results: There was no significant difference between the two drugs in decreasing erythema, swelling, crust, scratch marks, lichenification, xerosis and pruritus (p = 1, 1, 0.82, 0.625, 0.761, 0.125, 0.54, respectively). Sertaconazole was significantly better in decreasing the total score (p = 0.023). In terms of patients' overall idea about the drugs, sertaconazole was significantly superior to hydrocortisone (p = 0.023). Conclusion: The study showed that sertaconazole was significantly better in decreasing the total score of the disease and patients' ideas. There was no significant difference between the two drugs regarding each sign by itself. Sertaconazole could be a safe and efficient treatment in AD.

Occurrence of Staphylococcus aureus and evaluation of anti-staphylococcal activity of Lactococcus lactis subsp. lactis in ready-to-eat poultry meat

A total of 181 ready-to-eat poultry meat samples were examined for the presence of Staphylococcus aureus, and 11 (6 %) were found to have S. aureus contamination. Of 11 S. aureus isolates, 10 (91 %) were resistant to at least one antibiotic used in this study, and 2 were resistant to oxacillin. Lactococcus lactis subsp. lactis was tested as a bio-control agent. All the S. aureus isolates were found to be sensitive to antimicrobial products in L. lactis subsp. lactis supernatants; the zones of inhibition were in the range of 5.0 mm ± 0.70 mm to 19.8 mm ± 0.83 mm with the majority of isolates. As a competitive flora in mixed culture (LAPTg broth) and protective culture in poultry meat, L. lactis subsp. lactis was effective against S. aureus isolates; the growth of S. aureus isolates was almost negative after 32 h incubation in mixed culture. The population of S. aureus was reduced substantially to almost log 1 CFU/g after 25 days of incubation in protective culture. The pH of the test cultures also decreased sharply with time.

Combined treatments of enterocin AS-48 with biocides to improve the inactivation of methicillin-sensitive and methicillin-resistant Staphylococcus aureus planktonic and sessile cells

Control of staphylococci during cleaning and disinfection is important to the food industry. Broad-spectrum bacteriocins with proved anti-staphylococcal activity, such as enterocin AS-48, could open new possibilities for disinfection in combination with biocides. In the present study, enterocin AS-48 was tested singly or in combination with biocides against a cocktail of six Staphylococcus aureus strains (including three methicillin-resistant strains) in planktonic state as well as in biofilms formed on polystyrene microtiter plates. Cells were challenged with enterocin, biocides or enterocin/biocide combinations. Inactivation of planktonic cells increased significantly (p<0.05) when enterocin AS-48 (25mg/l) was tested in combination with benzalkonium chloride (BC), cetrimide (CT) and hexadecylpyridinium chloride (HDP), and non-significantly in combination with didecyldimethylammonium bromide (AB), triclosan (TC), hexachlorophene (CF), polyhexamethylen guanidinium chloride (PHMG), chlorhexidine (CH) or P3-oxonia (OX). In the sessile state (24h biofilms), staphylococci required higher biocide concentrations in most cases, except for OX. Inactivation of sessile staphylococci increased remarkably when biocides were applied in combination with enterocin AS-48, especially when the bacteriocin was added at 50mg/l. During storage, the concentrations of sessile as well as planktonic cells in the treated samples decreased remarkably for BC, TC and PHMG, but OX failed to inhibit proliferation of the treated biofilms as well as growth of planktonic cells. The observed inhibitory effects during storage were potentiated when the biocides were combined with 50mg/l enterocin AS-48. Results from this study suggest that selected combinations of enterocin AS-48 and biocides offer potential use against planktonic and sessile, methicillin-sensitive and methicillin-resistant S. aureus.