Mirjana Skočibušić

Phytochemical composition and antimicrobial activities of the essential oils from Satureja subspicata Vis. growing in Croatia

The present study describes the phytochemical profile and antimicrobial activity of Satureja subspicata Vis. essential oils, collected in Dalmatia (Croatia). Three samples of essential oils were obtained from the aerial parts of the plant by hydrodistillation and analyzed by GC-MS. From the twenty-four compounds representing 97.47% of the oils, carvacrol (16.76%), α -pinene (13.58), p-cymene (10.76%), γ -terpinene (9.54%) and thymol methyl ether (8.83%) appear as the main components. The oils also contained smaller percentages of myrcene, linalool, β -caryophyllene, limonene, geranial acetate, 1-Octen-3-ol, nerol, thymol and borneol. Furthermore, antimicrobial activity of the oil was evaluated using agar diffusion and broth microdilution methods. The antimicrobial test results showed that the oils had a great potential antimicrobial activity against all 13 bacteria and 9 fungal strains. Gram-positive bacteria are more sensitive to the investigated oil, with a range of 0.09 to 6.25 µ ; ; l/ml than Gram-negative bacteria in the range which is significantly higher from 1.56 to 25.00 µ ; ; l/ml. Results presented here may suggest that the essential oil of S. subspicata possess antimicrobial properties, and is therefore a potential source of antimicrobial ingredient in food and pharmaceutical industry.

Chemical Composition and Antidiarrhoeal Activities of Winter Savory (Satureja montana L.) Essential Oil

The purpose of this study was to examine chemical composition and the effects of Satureja montana L. (Lamiaceae) essential oil on the important enteric bacterial pathogens such as diarrheagenic Escherichia coli, Plesiomonas shigelloides, Shigella flexneri, Salmonella enterica serovar typhimurium, Yersinia enterocolitica and Vibrio parahaemolyticus. Leaves and flowers of the investigated plant were collected from a population growing in the littoral part of Dalmatia (Croatia). The GC/MS analyses revealed 33 compounds, the monoterpenes being the major fraction. Among them, the oxygen-containing phenolic monoterpene carvacrol (50.2%) and thymol (11%) were found to be the main constituents. The antibacterial activity of essential oil was evaluated by a modified agar dilution assay. The inhibitory properties of essential oils were observed within the range from 0.02 to 0.64%. Maximum activity was observed against Sh. flexneri and E. coli. Our results suggest that the essential oil of S. montana may warrant further investigation for their potential therapeutic efficacy in enteric tract infections.

Prevalence and diversity of extended-spectrum-β-lactamase-producing Enterobacteriaceae from marine beach waters

A total of 1,351 Enterobacteriaceae isolates from 144 seawater samples were collected over a four-year period from three public beaches in the eastern Adriatic Sea in Croatia. Approximately 35% of the strains were multidrug-resistant. BlaESBL genes were detected in 4.2% of the isolated Enterobacteriaceae, the main species of which were Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae. BlaTEM-1+SHV-12 was the most dominant genotype, followed by blaCTX-M-15.Raoultella terrigena and E. intermedius simultaneously harboured blaTEM-1,blaSHV-11/12 and blaCTX-M-15. Isolate fingerprinting revealed that marine E. coli isolates were clonally related to CTX-M-producing strains from a regional university hospital. These results indicate that marine beach waters are reservoirs of ESBL-producing Enterobacteriaceae and thus constitute a public health problem with further potential to act as mediators in gene flow between marine coastal areas and clinical settings.

Aeromonas spp. simultaneously harbouring blaCTX-M-15, blaSHV-12, blaPER-1 and blaFOX-2, in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia

Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired β-lactamases, including extended-spectrum-β-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel (Mytilus galloprovincialis) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and β-lactam/β-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC (bla(FOX), bla(CMY), bla(MOX), bla(LAT), bla(BIL), bla(DHA), bla(ACC), bla(MIR), bla(ACT)), ESBLs (bla(TEM), bla(SHV), bla(CTX-M), bla(PER), bla(VEB), bla(GES/IBC), bla(OXA)) and integrases (intI1, intI2, intI3). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila) isolates, with bla(CTX-M-15) gene identified in 19 and bla(SHV-12) in 12 isolates. Among them, 10 isolates simultaneously harboured bla(CTX-M-15) and bla(SHV-12), while 3 isolates additionally carried an AmpC β-lactamase bla(FOX-2) gene. bla(PER-1) gene was identified in a single isolate also harbouring the bla(CTX-M-15) gene. While bla(SHV-12) was chromosomally encoded, bla(CTX-M-15) was located on conjugative IncFIB-type plasmids of ~40 kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates. To the best our knowledge, this is the first report of environmental A. caviae isolates producing CTX-M-15, and isolation of SHV-12-producing A. hydrophila and A. caviae strains worldwide. This is also believed to be the first report of the FOX-2, CTX-M-15 and SHV-12 simultaneous production in aeromonads, highlighting both the potential risk for human health, and a role of these foodborne pathogens as reservoirs of resistance determinants in coastal marine environment.

Phytochemical Analysis and Antimicrobial Activity of Cardaria draba (L.) Desv. Volatiles

Two different volatile isolates from the aerial parts of Cardaria draba (L.) Desv., obtained either by hydrodistillation (Extract I) or by CH2Cl2 extraction subsequent to hydrolysis by exogenous myrosinase (Extract II), were characterized by GC‐FID and GC/MS analyses. The main volatiles obtained by hydrodistillation, i.e., 4‐(methylsulfanyl)butyl isothiocyanate (1; 28.0%) and 5‐(methylsulfanyl)pentanenitrile (2; 13.8%), originated from the degradation of glucoerucin. In Extract I, also volatiles without sulfur and/or nitrogen were identified. These were mostly hexadecanoic acid (10.8%), phytol (10.2%), dibutyl phthalate (4.5%), and some other compounds in smaller percentages. Extract II contained mostly glucosinolate degradation products. They originated from glucoraphanin, viz., 4‐(methylsulfinyl)butyl isothiocyanate (3; 69.2%) and 5‐(methylsulfinyl)pentanenitrile (4; 4.5%), glucosinalbin, viz., 2‐(4‐hydroxyphenyl)acetonitrile (5; 7.2%), and glucoerysolin, viz., 4‐(methylsulfonyl)butyl isothiocyanate (6; 5.0%). Moreover, the volatile samples were evaluated for their antimicrobial activity using the disc‐diffusion method and determining minimum inhibitory concentrations (MIC). All volatile isolates expressed a wide range of growth inhibition activity against both Gram‐positive and Gram‐negative bacteria and fungi. The MIC values varied between 4 and 128 μg/ml.

Synthesis and antimicrobial profile of N-substituted imidazolium oximes and their monoquaternary salts against multidrug resistant bacteria

Two different series of N-substituted imidazolium oximes and their monoquaternary salts were synthesized and biologically tested with respect to their ability to inhibit growth a diverse panel of antibiotic susceptible Gram-positive and antibiotic resistant Gram-negative bacteria as well fungal strains. The newly synthesized compounds were analyzed by spectral studies to confirm their structure. The preliminary results showed that all compounds tested possess promising antimicrobial potential against both susceptible Gram-positive and antibiotic resistant Gram-negative isolates, exhibiting a wide range of MIC values from 0.14 to 100.0 μg/mL. The structure-activity relationship demonstrates that the p-methylphenyl and p-fluorophenyl groups in monoquaternary salts 6 and 7 attached directly to the imidazolium ring could be essential for observed remarkable inhibitory profiles against clinically important pathogens Pseudomonas aeruginosa (MIC=0.14 μg/mL) and Klebsiella pneumoniae (MIC=1.56 μg/mL). Furthermore, the broth microdilution assay was then used to investigate the antiresistance efficacy of compound 7 against fourteen extended-spectrum β-lactamase (ESBL)-producing strains in comparison to eight clinically relevant antibiotics. Compound 7 exhibited a remarkable antiresistance profiles ranging between 0.39 and 12.50 μg/mL against all of ESBL-producing strains, which leads to the suggestion that may be interesting candidate for development of new antimicrobials to combat multidrug resistant Gram-negative bacteria.

Chemical composition and antimicrobial activity of the essential oil of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica).

The chemical composition and the antimicrobial activity of the essential oil isolated from the needles of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica) from Croatia were investigated. The chemical composition of the essential oil was determined by GC and GC/MS analyses, and the main compounds identified were α‐pinene, β‐pinene, germacrene D, and β‐caryophyllene. Disc‐diffusion and broth‐microdilution assays were used for the in vitro antimicrobial screening. The Dalmatian black pine essential oil exhibited a great potential of antibacterial activity against Gram‐positive bacteria (MIC=0.03–0.50% (v/v)) and a less pronounced activity against Gram‐negative bacteria (MIC=0.12–3.2% (v/v)). The volatile compounds also inhibited the growth of all fungi tested, including yeast.

Hedge Mustard (Sisymbrium officinale): Chemical Diversity of Volatiles and Their Antimicrobial Activity

Volatile compounds of hedge mustard (Sysimbrium officinale) have been investigated for the first time. Forthy‐two compounds were identified after hydrodistillation (without or upon autolysis) after gas chromatography and gas chromatography/mass spectrometry analyses. In addition, after decoction and hydrolysis of O‐glycosides, 18 volatile O‐aglycones were identified. In general, the main volatiles found in hydrodistillates were: isopropyl isothiocyanate (27.6–48.9%), 2‐methylpropanenitrile (0.5–18.8%), (Z)‐hex‐3‐en‐1‐ol (0.5–18.0%), sec‐butyl isothiocyanate (4.9–9.4%), (E)‐hex‐2‐enal (3.5–8.6%), (Z)‐hex‐2‐en‐1‐ol (0.3–8.4%), octanoic (0.5–8.6%) and dodecanoic acid (0–5.0%), 2‐methylbutanenitrile (0–4.6%), dibutyl phthalate (0–4.5%), and ethyl linolenate (0–3.6%). The main volatile O‐aglycones were: 2‐phenylethyl alcohol (21.5%), 6,7‐dehydro‐7,8‐dihydro‐3‐oxo‐α‐ionol (9.3%), eugenol (8.3%), benzyl alcohol (7.0%), ethyl vanillate (5.2%), 6‐(tert‐butyl)‐5‐methylphenol (5.1%), vanillin acetone (4.7%), ethyl 4‐hydroxybenzoate (4.3%), and 2‐hydroxy‐β‐ionone (3.8%). All hydrodistillates exhibited great potential of antibacterial activity against five Gram‐positive bacteria, nine ampicillin‐resistant Gram‐negative bacteria, and four fungi at a concentration of 500 μg/ml using the disc diffusion method.

Glucosinolate Profiling and Antimicrobial Screening of Aurinia leucadea (Brassicaceae)

Glucosinolates (GLs) were characterized in various aerial parts (stems, leaves, and flowers) of Aurinia leucadea (Guss.) C. Koch and quantified according to the ISO 9167‐1 official method based on the HPLC analysis of desulfoglucosinolates. Eight GLs, i.e., glucoraphanin (GRA), glucoalyssin (GAL; 1), gluconapin (GNA; 2), glucocochlearin (GCC), glucobrassicanapin (GBN; 3), glucotropaeolin (GTL), glucoerucin (GER), and glucoberteroin (GBE) were identified. The total GL contents were 57.1, 37.8, and 81.3 μmol/g dry weight in the stems, leaves, and flowers, respectively. The major GL detected in all parts of the plant was 2, followed by 1 and 3. GC/MS Analysis of the volatile fractions extracted from the aerial parts of fresh plant material either by hydrodistillation or CH2Cl2 extraction showed that these fractions mostly contained isothiocyanates (ITCs). The main ITCs were but‐3‐enyl‐ (55.6–71.8%), pent‐4‐enyl‐ (7.6–15.3%), and 5‐(methylsulfinyl)pentyl ITC (0–9.5%), originating from the corresponding GLs 2, 3, and 1, respectively. The antimicrobial activity of the volatile samples was investigated by determining inhibition zones with the disk‐diffusion method and minimal inhibitory concentrations (MIC) with the microdilution method. They were found to inhibit a wide range of bacteria and fungi, with MIC values of 2.0–32.0 μg/ml, indicating their promising antimicrobial potential, especially against the fungi Candida albicans and Rhizopus stolonifer as well as against the clinically important pathogen Pseudomonas aeruginosa.

Characterization of Environmental CTX-M-15-Producing Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a widespread environmental microorganism (1) that has emerged as significant opportunistic pathogen (2) due to intrinsic resistance to almost all available antibiotics.…