3-Phenyl substituted 6,7-dimethoxyisoquinoline derivatives as FtsZ-targeting antibacterial agents

The emergence of multidrug-resistant bacteria has created an urgent need for antibiotics with a novel mechanism of action. The bacterial cell division protein FtsZ is an attractive target for the development of novel antibiotics. The benzo[c]phenanthridinium sanguinarine and the dibenzo[a,g]quinolizin-7-ium berberine are two structurally similar plant alkaloids that alter FtsZ function. The presence of a hydrophobic functionality at either the 1-position of 5-methylbenzo[c]phenanthridinium derivatives or the 2-position of dibenzo[a,g]quinolizin-7-ium derivatives is associated with significantly enhanced antibacterial activity. 3-Phenylisoquinoline represents a subunit within the ring-systems of both of these alkaloids. Several 3-phenylisoquinolines and 3-phenylisoquinolinium derivatives have been synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, including multidrug-resistant strains of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). A number of derivatives were found to have activity against both MRSA and VRE. The binding of select compounds to S. aureus FtsZ (SaFtsZ) was demonstrated and characterized using fluorescence spectroscopy. In addition, the compounds were shown to act as stabilizers of SaFtsZ polymers and concomitant inhibitors of SaFtsZ GTPase activity. Toxicological assessment of select compounds revealed minimal cross-reaction mammalian β-tubulin as well as little or no human cytotoxicity.     

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.     

Persistence of Vancomycin-Resistant Enterococci in New Zealand Broilers after Discontinuation of Avoparcin Use

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was ≥256 μg/ml for 98.7% of isolates, and the avilamycin MIC was ≥8 μg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.     

Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources

Aim: Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin‐resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production. Methods and Results: Among the isolates, 27 (8·9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against Gram‐positive and some Gram‐negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS‐48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL_L gene in eight. Among the strains showing the cylL_L gene, only two E. faecalis expressed a β‐haemolysis. Conclusions: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. Significance and Impact of the Study: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits.     

Activity and synergistic interactions of stilbenes and antibiotic combinations against bacteria in vitro

The synergistic antibacterial activity of two stilbenes [3,4??,5-trihydroxystilbene (1) and 3,5-dihydroxy-4-isopropylstilbene(2)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) in combination with ciprofloxacin and cefotaxime was investigated. The activity of the stilbenes and standard antibiotics on bacteria were determined using the macrodilution method. The minimum inhibitory concentration and minimum bactericidal concentration of the stilbenes was compared with that of the standard antibiotics. The antibacterial activities of stilbenes against pathogenic bacteria were assessed using the checkerboard and time-kill methods to evaluate the synergistic effects of stilbenes in treatment with ciprofloxacin or cefotaxime. The results of the present study showed that the combination effects of both stilbenes with ciprofloxacin were synergistic (FIC index <0.5). Whereas stilbene 2 with cefotaxime recorded additive. Furthermore, time-kill study showed that the growth of the tested bacteria was completely attenuated with 12?C24?h of treatment with 50:50 ratios of stilbenes and antibiotics. These results suggest that stilbenes combined with antibiotics may be microbiologically beneficial and not antagonistic. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and stilbenes). The in vitro synergistic activity of stilbenes and antibiotics against bacteria is reported here for the first time.     

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides

One of the promising directions of the combined approach is the design of dual-acting antibiotics – heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin’s activity for the treatment of mice with Staphylococcus aureus sepsis.     

Biological evaluation and molecular modelling study of thiosemicarbazide derivatives as bacterial type IIA topoisomerases inhibitors

In the present article, we describe the inhibitory potency of nine thiosemicarbazide derivatives against bacterial type IIA topoisomerases, their antibacterial profile and molecular modelling evaluation. We found that one of the tested compounds, compound 7, significantly inhibits activity of Staphylococcus aureus DNA gyrase with an IC₅₀ below 15?μM. Besides, this compound displays antibacterial activity on reference Staphylococuss spp. and Enterococcus faecalis strains as well as clinical S. aureus isolates at non-cytotoxic concentrations in mammalian cells with MIC values ranging from 16 to 32?μg/mL thereby indicating, in some cases, equipotent or even more effective action than standard drugs such as vancomycin, ampicillin and nitrofurantoin. The computational studies showed that both molecular geometry and the electron density distribution have a great impact on antibacterial activity of thiosemicarbazide derivatives.     

Pharmacological synergism of bee venom and melittin with antibiotics and plant secondary metabolites against multi-drug resistant microbial pathogens

The goal of this study was to investigate the antimicrobial activity of bee venom and its main component, melittin, alone or in two-drug and three-drug combinations with antibiotics (vancomycin, oxacillin, and amikacin) or antimicrobial plant secondary metabolites (carvacrol, benzyl isothiocyanate, the alkaloids sanguinarine and berberine) against drug-sensitive and antibiotic-resistant microbial pathogens. The secondary metabolites were selected corresponding to the molecular targets to which they are directed, being different from those of melittin and the antibiotics.The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were evaluated by the standard broth microdilution method, while synergistic or additive interactions were assessed by checkerboard dilution and time-kill curve assays. Bee venom and melittin exhibited a broad spectrum of antibacterial activity against 51 strains of both Gram-positive and Gram-negative bacteria with strong anti-MRSA and anti-VRE activity (MIC values between 6 and 800 µg/ml). Moreover, bee venom and melittin showed significant antifungal activity (MIC values between 30 and 100 µg/ml). Carvacrol displayed bactericidal activity, while BITC exhibited bacteriostatic activity against all MRSA and VRE strains tested (reference strains and clinical isolates), both compounds showed a remarkable fungicidal activity with minimum fungicidal concentration (MFC) values between 30 and 200 µg/ml. The DNA intercalating alkaloid sanguinarine showed bactericidal activity against MRSA NCTC 10442 (MBC 20 µg/ml), while berberine exhibited bacteriostatic activity against MRSA NCTC 10442 (MIC 40 µg/ml).Checkerboard dilution tests mostly revealed synergism of two-drug combinations against all the tested microorganisms with FIC indexes between 0.24 and 0.50, except for rapidly growing mycobacteria in which combinations exerted an additive effect (FICI = 0.75–1). In time-kill assays all three-drug combinations exhibited a powerful bactericidal synergistic effect against MRSA NCTC 10442, VRE ATCC 51299, and E. coli ATCC 25922 with a reduction of more than 3log₁₀ in the colony count after 24 h. Our findings suggest that bee venom and melittin synergistically enhanced the bactericidal effect of several antimicrobial agents when applied in combination especially when the drugs affect several and differing molecular targets. These results could lead to the development of novel or complementary antibacterial drugs against MDR pathogens.     

Synthesis and Antibacterial Activity Evaluation of Biphenyl and Dibenzofuran Derivatives as Potential Antimicrobial Agents against Antibiotic-Resistant Bacteria

The escalating prevalence of antibiotic-resistant bacteria has led to a serious global public health problem; therefore, there is an urgent need for the development of structurally innovative antibacterial agents. In our study, a series of biphenyl and dibenzofuran derivatives were designed and synthesized by Suzuki-coupling and demethylation reactions in moderate to excellent yields (51–94% yield). Eleven compounds exhibited potent antibacterial activities against the prevalent antibiotic-resistant Gram-positive and Gram-negative pathogens, among which compounds 4′-(trifluoromethyl)-[1,1′-biphenyl]-3,4,5-triol (6i) and 5-(9H-carbazol-2-yl) benzene-1,2,3-triol (6m) showed the most potent inhibitory activities against methicillin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with MIC (minimum inhibitory concentration) values as low as 3.13 and 6.25 μg/mL, respectively. Compounds 3′,5′-dimethyl-[1,1′-biphenyl]-3,4,4′,5-tetraol (6e), 4′-fluoro-[1,1′-biphenyl]-3,4,5-triol (6g), and 4′-(trifluoromethyl)-[1,1′-biphenyl]-3,4,5-triol (6i) showed comparable inhibitory activities with ciprofloxacin to Gram-negative bacterium carbapenems-resistant Acinetobacter baumannii. Study of the structure–activity relationship indicated that a strong electron-withdrawing group on the A ring and hydroxyl groups on the B ring of biphenyls were beneficial to their antibacterial activities, and for benzo-heterocycles, N-heterocycle exhibited optimal antibacterial activity. These results can provide novel structures of antibacterial drugs chemically different from currently known antibiotics and broaden prospects for the development of effective antibiotics against antibiotic-resistant bacteria.     

Design,synthesis and biological evaluation of spiropyrimidinetriones oxazolidinone derivatives as antibacterial agents

Gram-positive bacteria are among the most common human pathogens associated with clinical infections which range from mild skin infections to sepsis. Resistance towards existing class of drugs by Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (MRSE) and vancomycin resistant enterococci (VRE) is a growing concern. There is an urgent need to discover new antibiotics which are active against resistant strains of Gram positive bacteria. We report herein a novel class of spiropyrimidinetrione oxazolidinone derivatives as novel antibacterial agents. Key step towards the synthesis of title compounds involved the use of tert-amino reaction with [1,5]-hydride shift leading to the new CC bond formation. Compound 30n has demonstrated potent antibacterial activity against a panel of Gram-positive microbial strains including MRSA, MRSE, and LNZ and vancomycin resistant strains of E. faecalis. Further, molecular docking studies suggest that 30n has binding mode similar to that of LNZ in 50S RNA ribosome.     

Nosocomial infection caused by vancomycin‐susceptible multidrug‐resistant Enterococcus faecalis over a long period in a university hospital in Japan

Compared with other developed countries, vancomycin‐resistant enterococci (VRE) are not widespread in clinical environments in Japan. There have been no VRE outbreaks and only a few VRE strains have sporadically been isolated in our university hospital in Gunma, Japan. To examine the drug susceptibility of Enterococcus faecalis and nosocomial infection caused by non‐VRE strains, a retrospective surveillance was conducted in our university hospital. Molecular epidemiological analyses were performed on 1711 E. faecalis clinical isolates collected in our hospital over a 6‐year period [1998–2003]. Of these isolates, 1241 (72.5%) were antibiotic resistant and 881 (51.5%) were resistant to two or more drugs. The incidence of multidrug resistant E. faecalis (MDR‐Ef) isolates in the intensive care unit increased after enlargement and restructuring of the hospital. The major group of MDR‐Ef strains consisted of 209 isolates (12.2%) resistant to the five drug combination tetracycline/erythromycin/kanamycin/streptomycin/gentamicin. Pulsed‐field gel electrophoresis analysis of the major MDR‐Ef isolates showed that nosocomial infections have been caused by MDR‐Ef over a long period (more than 3 years). Multilocus sequence typing showed that these strains were mainly grouped into ST16 (CC58) or ST64 (CC8). Mating experiments suggested that the drug resistances were encoded on two conjugative transposons (integrative conjugative elements), one encoded tetracycline‐resistance and the other erythromycin/kanamycin/streptomycin/gentamicin‐resistance. To our knowledge, this is the first report of nosocomial infection caused by vancomycin‐susceptible MDR‐Ef strains over a long period in Japan.     

Synthesis and antibacterial activity study of a novel class of cationic anthraquinone analogs

Reported previously by our group, one-pot cycloaddition using naphthoquinone, sodium azide and alkyl halides can lead to the formation of both 1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones. Herein, the effect of leaving group and additive in dictating the selectivity between the formation of 1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones has been further investigated. In the process of investigating the factors that control the selectivity and the biological activity associated with these two compounds, a novel class of antibacterial cationic anthraquinone analogs has been developed. Although these compounds are structurally similar, different antibacterial profiles are noted. One lead compound, 4e manifests high potency (MIC < 1 ??g/mL) and selectivity against Gram positive (G+) pathogens including methicillin-resistant Staphylococcus aureus (MRSA) while exerting only modest activity against Gram negative (G−) bacteria. Other lead compounds (4f and 4g) exhibit broad antibacterial activity including MRSA and vancomycin-resistant Enterococcus faecalis (VRE) that is comparable to other commercially available cationic antiseptic chemicals. This unique difference in antibacterial profile may pave the way for the development of new therapeutic agents.     

New fluorescent rosamine chelator showing promising antibacterial activity against Gram-positive bacteria

The restricted number of antibiotics to treat infections caused by common multidrug resistant bacterial pathogens in the clinical setting demands a continuous search for new molecules with antibacterial properties. Bacterial iron deprivation represents a promising alternative, being iron chelators an attractive class for drug design in which particular compounds seem to have antibacterial effect.In this work, we report the synthesis and characterization of a new fluorescent 3-hydroxy-4-pyridinone (3,4-HPO) iron chelator functionalized with a carboxyrosamine fluorophore (MRB20). The antibacterial activity of MRB20 was assessed against representative strains from clinically relevant Gram-positive and Gram-negative bacterial species and further compared with the inhibitory effect of a set of structurally related iron chelators including Deferiprone (1,2-dimethyl-3-hydroxy-4-pyridinone). Compounds exhibiting a promising minimal inhibitory concentration (MIC < 10 mg/L) were further tested against a wider range of bacterial genera and species (Staphylococcus spp. Enterococcus spp. Listeria monocytogenes, Bacillus spp.), including multidrug resistant bacteria.With the exception of the novel compound (MRB20), all chelators inhibited the strains assayed at very high concentrations [minimum inhibitory concentrations (MIC) ranging from 70 mg/L to >180 mg/L]. MRB20 revealed a good antibacterial activity (6.7–13.2 mg/L) against Gram-positive strains from different genera and species, including clinically relevant species (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis), which might be eventually compatible with a therapeutic application or as adjuvant.     

Synthesis and in vitro activity of novel isoxazolyl tetrahydropyridinyl oxazolidinone antibacterial agents

A series of isoxazolyl tetrahydropyridinyl oxazolidinones with various substituents at the 3-position of the isoxazole ring have been synthesized and their in vitro antibacterial activities (MIC) were evaluated against several Gram-positive strains including the resistant strains of Staphyloccus and Enterococcus, such as MRSA and VRE. One of the most potent compounds synthesized, 4f, showed comparable or better activity against selected bacterial strains than those of linezolid and vancomycin.     

Antibacterial Activity of THAM Trisphenylguanide against Methicillin-Resistant Staphylococcus aureus

This study investigated the potential antibacterial activity of three series of compounds synthesized from 12 linear and branched polyamines with 2–8 amino groups, which were substituted to produce the corresponding guanides, biguanides, or phenylguanides, against Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Antibacterial activity was measured for each compound by determining the minimum inhibitory concentration against the bacteria, and the toxicity towards mammalian cells was determined. The most effective compound, THAM trisphenylguanide, was studied in time-to-kill and cytoplasmic leakage assays against methicillin-resistant Staphylococcus aureus (MRSA, USA300) in comparison to chlorhexidine. Preliminary toxicity and MRSA challenge studies in mice were also conducted on this compound. THAM trisphenylguanide showed significant antibacterial activity (MIC ∼1 mg/L) and selectivity against MRSA relative to all the other bacteria examined. In time-to-kill assays it showed increased antimicrobial activity against MRSA versus chlorhexidine. It induced leakage of cytoplasmic content at concentrations that did not reduce cell viability, suggesting the mechanism of action may involve membrane disruption. Using an intraperitoneal mouse model of invasive MRSA disease, THAM trisphenylguanide reduced bacterial burden locally and in deeper tissues. This study has identified a novel guanide compound with selective microbicidal activity against Staphylococcus aureus, including a methicillin-resistant (MRSA) strain.     

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10⁻⁶ per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.     

News & Notes: Growth Inhibition of Pseudomonas aeruginosa and Enterococcus faecalis by Crude Extractives of Mansonia altisima Timber Sawdust

Crude, aqueous methanol and ethanol extracts of sawdust of Mansonia (Mansonia altisima) timber inhibited Pseudomonas aeruginosa and Enterococcus faecalis. The minimum inhibitory concentration ranged from 4.4 to 9.3 mg/ml, while the minimum bactericidal concentration was 10.0–25.0 mg/ml. Enterococcus faecalis was more susceptible, but no marked difference occurred in the antibacterial effect of methanol and ethanol extracts. The aqueous extract was less active.     

Substituted 1,6-diphenylnaphthalenes as FtsZ-targeting antibacterial agents

Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multidrug-resistant strains of infectious bacteria. Several 1-phenylbenzo[c]phenanthridines exhibit notable antibacterial activity. Based upon their structural similarity to these compounds, a distinct series of substituted 1,6-diphenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. In addition, the effect of select 1,6-diphenylnaphthalenes on the polymerization dynamics of S. aureus FtsZ and mammalian β-tubulin was also assessed. The presence of a basic functional group or a quaternary ammonium substituent on the 6-phenylnaphthalene was required for significant antibacterial activity. Diphenylnaphthalene derivatives that were active as antibiotics, did exert a pronounced effect on bacterial FtsZ polymerization and do not appear to cross-react with mammalian tubulin to any significant degree.     

In Vitro Evaluation of the Risk of Inducing Bacterial Resistance to Disinfection Treatment with Photolysis of Hydrogen Peroxide

The purpose of the present study was to evaluate the risk of inducing bacterial resistance to disinfection treatment with photolysis of H₂O₂ and comparing this with existing antibacterial agents. We tested seven antibacterial agents, including amoxicillin, cefepime hydrochloride, erythromycin, ofloxacin, clindamycin hydrochloride, ciprofloxacin hydrochloride, and minocycline hydrochloride, as positive controls for validation of the assay protocol. For all of the agents tested, at least one of the four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Streptococcus salivarius) was resistant to these agents by repeated exposure to subinhibitory concentrations of the agents up to 10 times. In contrast, antibacterial activity against any of the bacterial species tested (S. aureus, E. faecalis, E. coli, S. salivarius, Pseudomonas aeruginosa, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans) was not affected by repeated exposure to the disinfection treatment up to 40 times. This finding suggested that the risk of inducing bacterial resistance by disinfection treatment was low. The active ingredient of this disinfection treatment is hydroxyl radicals generated by photolysis of H₂O₂. Therefore, hydroxyl radicals interact with several cell structures and different metabolic pathways in microbial cells, probably resulting in a lack of development of bacterial resistance. In conclusion, disinfection treatment with photolysis of H₂O₂ appears to be a potential alternative for existing antimicrobial agents in terms of a low risk of inducing bacterial resistance.     

Bacteriocinogenic properties and in vitro probiotic potential of enterococci from Tunisian dairy products

The aim of this study was to isolate new bacteriocinogenic strains with putative probiotic potential from various Tunisian fermented milks. A total of 44 Gram-positive catalase-negative isolates were colony-purified and screened for antimicrobial activity. Of inhibitory isolates, four were identified as Enterococcus durans and one as Enterococcus faecalis using 16S rRNA gene sequence. The five strains were sensitive to penicillin G, all aminoglycosides tested, to the vancomycin, tetracycline, and chloramphenicol, and E. durans 42G and E. faecalis 61B were resistant to erythromycin. The antimicrobial substances were sensitive to proteolytic enzymes and had good biochemical stability. E. durans 61A showed a good resistance to gastric and small intestinal secretions, but were more sensitive to the duodenal conditions. Considering the safety and the stability under simulated gastrointestinal tract, it appears that the bacteriocinogenic strain E. durans 61A is a good candidate for its application as novel probiotic strain in the food industry.