Influence of essential oil Helichrysum italicum (Roth) G. Don on the formation of non-tuberculous mycobacterial biofilm

Cilj: Ispitati antimikrobni i antioksidacijski učinak te sposobnost inhibicije biofilma eteričnog ulja (EU) smilja [Helichrysum italicum (Roth) G. Don (H. italicum)] prema netuberkuloznim mikobakterijama M. avium i M. intracellulare. Materijali i metode: Za određivanje minimalne inhibicijske koncentracije (MIK) i minimalne baktericidne koncentracije (MBK) EU H. italicum prema M. avium i M. intracellulare korištena je metoda mikrodilucije u bujonu. Preživljavanje mikobakterija u Middlebrook 7H9 bujonu pod utjecajem EU smilja praćeno je subkultivacijom suspenzija 0, 1, 4 i 8 dan. Za određivanje inhibicije stvaranja biofilma u vodi primjenjena je metoda bojenja biofilma s kristal violetom. Antioksidacijski učinak MIK koncentracije EU određen je DPPH (2,2-difenil-1-pikril-hidrazil) metodom. Rezultati: MIK i MBK vrijednost EU smilja iznosila je 3,2 mg/ml. Koncentracija ½ MIK vrijednosti dovela je nakon prvog dana do redukcije vijabilnosti M. avium za približno 2 log10 odnosno 2,5 log10 za M. intracellulare, dok je četvrti dan zabilježena potpuna inhibicija porasta obje vrste mikobakterija. M. avium je pokazao veću tendenciju stvaranja biofilma. Koncentracija od ¼ i ½ MIK vrijednosti dovela je do statistički značajne inhibicije stvaranja biofilma ispitivanih mikobakterija. MIK koncentracija EU H. italicum je nakon 60 minuta inhibirala 38,2 % DPPH radikala. Zaključak: Subinhibitorne koncentracije EU smilja značajno reduciraju vijabilnost M. avium i M. intracellulare i dovode do značajne inhibicije stvaranja biofilma ispitivanih mikobakterija u vodi što bi omogućilo primjenu niskih efektivnih koncentracija ovog EU, koje nemaju citotoksični učinak, kao prirodnog dezinficijensa u vodi.Objective: To investigate the antimicrobial and antioxidative effect of the essential oil (EO) of immortelle [Helichrysum italicum (Roth) G. Don (H. italicum)] and its ability to inhibit biofilm of nontuberculous mycobacteria, M. avium and M. intracellulare. Materials and methods: a broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the H. italicum EO. Survival of mycobacteria in Middlebrook 7H9 broth under the influence of H. italicum EO was monitored by subcultivation of suspension of 0, 1, 4 and 8 days. For determination of the inhibition of biofilm formation in water, the biofilm coloring method with crystal violet was applied. The antioxidant effect of MIC concentrations of H. italicum EO was determined by DPPH (2,2-diphenyl-1-picrylhydrazil) method. Results: MIC and MBC value of immortelle EO was 3.2 mg/ml. Treatment of mycobacteria with H. italicum EO at one half times the MIC resulted in approximately 2 log10 reduction of M. avium and 2.5 log10 M. intracellulare respectively over 24 h, and complete reduction of both mycobacteria on the fourth day. M. avium showed a greater tendency to form biofilms. The concentration of one-quarter and one-half of the MIC values resulted in statistically significant inhibition of biofilm production of the tested mycobacteria. MIC concentrations of H. italicum EU after 60 minutes inhibited 38.2% of DPPH radicals. Conclusion: The subinhibitory concentrations of H. italicum EO significantly reduce the viability of M. avium and M. intracellulare and result in substantial inhibition of biofilm production of the tested mycobacteria in water, which would allow the application of low effective concentrations of this EO that do not have a cytotoxic effect as a natural disinfectant in water

Synergistic potential of Juniperus communis and Helichrysum italicum essential oils against nontuberculous mycobacteria

Objective. The present study evaluated the possible synergistic antimycobacterial interactions of Juniperus communis (J. communis) and Helichrysum italicum (H. italicum) essential oils (EO). Methods. Antimycobacterial potential was tested against Mycobacterium avium and Mycobacterium intracellulare using broth and water dilution method and checkerboard synergy method. Antiadhesion and antibiofilm effect of EOs was evaluated on biotic (HeLa cells) and abiotic surface (polystyrene). To evaluate the possible mechanisms of action, cellular leakage of proteins and DNA was tested and structural changes were visualized with a transmission electron microscope. Results. MIC, MBC and MEC were 1.6 mg/ml for J. communis EO and 3.2 mg/ml for H. italicum EO against both mycobacteria. All combinations of EOs in checkerboard synergy method produced fractional inhibitory concentration index values ranging from 0.501 to 1.5, corresponding to synergistic, additive or indifferent effects. Mycobacterium avium showed a greater tendency to create biofilm but these EO at subinhibitory concentrations (sMIC) effectively blocked the adhesion and the establishment of biofilm. The exposure of both mycobacteria to MICs and sMICs lead to significant morphological changes: acquired a swollen form, ghost-like cell, disorganized cytoplasm detached from the cell wall. OD value of supernatant for both mycobacteria exposed to EOs have confirmed that there is a leakage of cellular material. Conclusion. The leakage of the cellular material is noticeably higher in sMIC, which is probably due to cell wall damage. sMIC of both EOs have an additive or synergistic effect, reducing MICs, limiting adhesion and preventing the formation of biofilms

Antimikobakterijski potencijal eteričnog ulja plodova borovice (Juniperus communis) u pitkoj vodi

Mycobacterium avium complex-related diseases are often associated with poorly maintained hot water systems. This calls for the development of new control strategies. The aim of this study was to investigate the activity of essential oils (EOs) from the Mediterranean plants, common juniper, immortelle, sage, lavandin, laurel, and white cedar against Mycobacterium avium ssp. avium, Mycobacterium intracellulare, and Mycobacterium gordonae in culturing broth and freshwater as their most common habitat. To do that, we developed a new method of water microdilution to determine their minimal effective concentrations (MEC). The most active EO was the one from the common juniper with the MEC of 1.6 mg mL-1. Gas chromatography / mass spectrometry the juniper EO identified monoterpenes (70.54 %) and sesquiterpenes (25.9 %) as dominant component groups. The main monoterpene hydrocarbons were α-pinene, sabinene, and β-pinene. The juniper EO significantly reduced the cell viability of M. intracellulare and M. gordonae at MEC, and of M. avium at 2xMEC. Microscopic analysis confirmed its inhibitory effect by revealing significant morphological changes in the cell membrane and cytoplasm of all three bacteria. The mode of action of the juniper EO on the cell membrane was confirmed by a marked leakage of intracellular material. Juniper EO has a great practical potential as a complementary or alternative water disinfectant in hot water systems such as baths, swimming pools, spa pools, hot tubs, or even foot baths/whirlpools.Ispitali smo aktivnost eteričnih ulja borovice [Juniperus communis (J. communis)], smilja (Helichrysum italicum), kadulje (Salvia officinalis), lavandina (Lavandula hybrida), lovora (Laurus nobilis) i tuje (Thuja occidentalis) prema sojevima Mycobacterium avium ssp. avium (M. avium), Mycobacterium intracellulare (M. intracellulare) i Mycobacterium gordonae (M. gordonae). Za određivanje minimalnih inhibicijskih koncentracija (MIK) i minimalnih baktericidnih koncentracija (MBK) testiranih eteričnih ulja koristili smo se metodom mikrodiluacije u tekućem bujonu (Middlebrook 7H9), a za određivanje minimalne efektivne koncentracije (MEK) umjesto bujona korištena je sterilna voda iz slavine kojoj je dodano 0,05 % Tweena 80. Dvostruka serijska razrjeđenja eteričnih ulja, počevši od 0,1 do 51,2 mg mL-1, u tekućem bujonu ili u sterilnoj vodi iz slavine inokulirana su s mikobakterijskom suspenzijom uz dodatak resazurina. Najučinkovitije eterično ulje prema svim ispitivanim sojevima mikobakterija bilo je eterično ulje J. communis s MBK / MIK / MEK vrijednošću od 1,6 mg mL-1. Metodom plinske kromatografije i masene spektrometrije analizirano je eterično ulje J. communis. Udio monoterpena iznosio je 70,54 %, a seskviterpena 25,9 %. Glavni monoterpenski ugljikovodici bili su α-pinen, sabinen i β-pinen. Eterično ulje J. communis pokazalo je značajan inhibicijski učinak na M. intracellulare i M. gordonae pri MEK i na M. avium pri dvostrukim MEK. Elektronskom mikroskopijom kod svih triju sojeva mikobakterija nakon izlaganja djelovanju eteričnog ulja J. communis otkrivene su značajne morfološke promjene stanične membrane i citoplazme. Učinak eteričnog ulja J. communis na destrukciju stanične membrane mikobakterija potvrđen je značajnim otpuštanjem unutarstaničnog materijala mjerenjem apsorbancije supernatanta pri 260 nm i 280 nm. Zaključno, u našem smo radu razvili novu metodu za ispitivanje antimikrobnog učinka eteričnih ulja ili drugih prirodnih tvari na netuberkulozne mikobakterije koja oponaša uvjete kao u vodenim sustavima. Prirodni proizvodi, osobito eterična ulja, imaju ne samo velik potencijal kao antimikrobni agensi nego i moguću praktičnu primjenu kao alternativni dezinficijensi

ANTIMICROBIAL EFFECT OF ESSENTIAL OILS ON NONTUBERCULOUS MYCOBACTERIA

Ciljevi istraživanja: Ispitati antimikrobni učinak eteričnih ulja (EU) odabranih mediteranskih biljaka (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis i Thuja occidentalis) i α-pinena na netuberkulozne mikobakterije (NTM) M. avium, M. intracellulare i M. gordonae te istražiti ulogu navedenih supstancija na biofilm NTM. Željeli smo utvrditi postojanje sinergističkih učinaka ispitivanih EU te kombinirane primjene EU i odabranih antimikrobnih lijekova i odrediti mehanizam djelovanja EU borovice na NTM. Metode: Uspostavom nove metode omogućeno je in vitro ispitivanje antimikrobnog učinka EU na NTM oponašajući uvjete vodoopskrbnih sustava. Određena je minimalna efektivna koncentracija (MEK) kao najmanja djelotvorna koncentracija EU u vodi. Metodom mikrodilucije u bujonu i steriliziranoj vodovodnoj vodi ispitana je osjetljivost NTM na odabrana EU i antimikrobne lijekove. Istražen je učinak inhibitornih i sinergističkih subinhibitornih koncentracija EU borovice i smilja na biofilm NTM na neživim površinama i površinama živih stanica. Mehanizam djelovanja EU borovice proučen je utvrđivanjem stupnja oštećenja stanične stijenke i morfoloških promijena primjenom transmisijskog elektronskog mikroskopa (TEM). Rezultati: EU borovice i smilja pokazala su se najučinkovitijima prema NTM, dok je α-pinen testiran samostalno pokazao slabiji učinak nego cjelovito EU. EU borovice iskazalo je u kombinaciji s EU smilja i tuje te antimikrobnim lijekovima niz sinergističkih učinaka prema NTM. Adhezija NTM na polistiren kao i formiranje biofilma, statistički značajno su inhibirani s porastom temperature i porastom koncentracije EU borovice. Sinergističke kombinacije EU borovice i antimikrobnih lijekova izazvale su povećano istjecanje staničnih makromolekula NTM. TEM uočene su promjene u obliku i strukturi NTM nakon djelovanja EU borovice i sinergističko djelovanje u kombinacijama s EU smilja. Zaključak: Pronašli smo niz sinergističkih kombinacija EU i antimikrobnih lijekova koje mogu imati potencijalnu primjenu u novim načinima sprječavanja adherencije i formiranja biofilma NTM ne samo u vodoopskrbnom sustavu, nego i na umjetnim materijalima koji se koriste u medicini te u biološkim sustavima. Sinergizam prilikom kombinirane primijene EU i antimikrobnih lijekova omogućuju primjenu niskih efektivnih koncentracija s održanim antimikrobnim učinkom ispitivanih supstancija s potencijalno smanjenom toksičnošću. EU borovice izaziva promjene u citoplazmi i oštećenje stanične stijenke NTM što je potvrđeno opaženim povećanim istjecanjem staničnih makromolekula i mikromorfološkim promjenama uočenim TEM.Research objectives: To investigate the antimicrobial effect of essential oils (EO) of selected Mediterranean plants (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis and Thuja occidentalis) and α-pinene on non-tuberculous mycobacteria (NTM), M. avium, M. intracelullare and M. gordonae and investigate the role of these substances on NTM biofilm. We wanted to determine the synergistic effects of the tested EO and combinations of EO and selected antimicrobials. The aim was also to determine the mechanism of action of common juniper EO on NTM. Methods: The establishment of a new method enabled the in vitro testing of the EO's antimicrobial effect on NTM by mimicking the conditions of water supply systems. The Minimum Effective Concentration (MEC) was determined as the lowest effective EO concentration in water. The sensitivity of NTM to selected EO and antimicrobials was examined by microdilution in broth and sterilized tap water. The effect of inhibitory and synergistic subinhibitory concentrations of common juniper EO and immortelle EO on biofilm of NTM on abiotic and biotic surfaces was investigated. The mechanism of action of common juniper EO has been studied by determining the degree of cell wall damage and morphological changes using a transmission electron microscope (TEM). Results: Common juniper and immortelle EO's proved to be most effective against NTM, while α-pinene tested alone showed poorer effect than overall EO. Common juniper EO have shown a number of synergistic effects, according to NTM, in combination with immortelle and white cedar EO and antimicrobials. The adhesion of NTM to polystyrene as well as biofilm formation were significantly inhibited (p < 0.05) with increasing temperature and increasing concentration of common juniper EO. Synergistic combinations of common juniper EO and antimicrobials have caused increased leakage of NTM cell macromolecules. TEM showed changes in the shape and structure of NTM after the activity of common juniper EO and synergistic effects in combination with immortelle EO. Conclusion: We have found a number of synergistic combinations of EO and antimicrobials that may have potential applications in new ways of preventing the adherence and formation of NTM biofilms not only in the water supply system, but also in artificial materials used in medicine and in biological systems. Synergies with the combined use of EO and antimicrobials allow the use of low effective concentrations with the sustained antimicrobial effect of the test substances with reduced toxicity. Common juniper EO cause changes in the cytoplasm and damage to the NTM cell wall, as evidenced by the observed increased leakage of cellular macromolecules and micromorphological changes observed by TEM

ANTIMICROBIAL EFFECT OF ESSENTIAL OILS ON NONTUBERCULOUS MYCOBACTERIA

Ciljevi istraživanja: Ispitati antimikrobni učinak eteričnih ulja (EU) odabranih mediteranskih biljaka (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis i Thuja occidentalis) i α-pinena na netuberkulozne mikobakterije (NTM) M. avium, M. intracellulare i M. gordonae te istražiti ulogu navedenih supstancija na biofilm NTM. Željeli smo utvrditi postojanje sinergističkih učinaka ispitivanih EU te kombinirane primjene EU i odabranih antimikrobnih lijekova i odrediti mehanizam djelovanja EU borovice na NTM. Metode: Uspostavom nove metode omogućeno je in vitro ispitivanje antimikrobnog učinka EU na NTM oponašajući uvjete vodoopskrbnih sustava. Određena je minimalna efektivna koncentracija (MEK) kao najmanja djelotvorna koncentracija EU u vodi. Metodom mikrodilucije u bujonu i steriliziranoj vodovodnoj vodi ispitana je osjetljivost NTM na odabrana EU i antimikrobne lijekove. Istražen je učinak inhibitornih i sinergističkih subinhibitornih koncentracija EU borovice i smilja na biofilm NTM na neživim površinama i površinama živih stanica. Mehanizam djelovanja EU borovice proučen je utvrđivanjem stupnja oštećenja stanične stijenke i morfoloških promijena primjenom transmisijskog elektronskog mikroskopa (TEM). Rezultati: EU borovice i smilja pokazala su se najučinkovitijima prema NTM, dok je α-pinen testiran samostalno pokazao slabiji učinak nego cjelovito EU. EU borovice iskazalo je u kombinaciji s EU smilja i tuje te antimikrobnim lijekovima niz sinergističkih učinaka prema NTM. Adhezija NTM na polistiren kao i formiranje biofilma, statistički značajno su inhibirani s porastom temperature i porastom koncentracije EU borovice. Sinergističke kombinacije EU borovice i antimikrobnih lijekova izazvale su povećano istjecanje staničnih makromolekula NTM. TEM uočene su promjene u obliku i strukturi NTM nakon djelovanja EU borovice i sinergističko djelovanje u kombinacijama s EU smilja. Zaključak: Pronašli smo niz sinergističkih kombinacija EU i antimikrobnih lijekova koje mogu imati potencijalnu primjenu u novim načinima sprječavanja adherencije i formiranja biofilma NTM ne samo u vodoopskrbnom sustavu, nego i na umjetnim materijalima koji se koriste u medicini te u biološkim sustavima. Sinergizam prilikom kombinirane primijene EU i antimikrobnih lijekova omogućuju primjenu niskih efektivnih koncentracija s održanim antimikrobnim učinkom ispitivanih supstancija s potencijalno smanjenom toksičnošću. EU borovice izaziva promjene u citoplazmi i oštećenje stanične stijenke NTM što je potvrđeno opaženim povećanim istjecanjem staničnih makromolekula i mikromorfološkim promjenama uočenim TEM.Research objectives: To investigate the antimicrobial effect of essential oils (EO) of selected Mediterranean plants (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis and Thuja occidentalis) and α-pinene on non-tuberculous mycobacteria (NTM), M. avium, M. intracelullare and M. gordonae and investigate the role of these substances on NTM biofilm. We wanted to determine the synergistic effects of the tested EO and combinations of EO and selected antimicrobials. The aim was also to determine the mechanism of action of common juniper EO on NTM. Methods: The establishment of a new method enabled the in vitro testing of the EO's antimicrobial effect on NTM by mimicking the conditions of water supply systems. The Minimum Effective Concentration (MEC) was determined as the lowest effective EO concentration in water. The sensitivity of NTM to selected EO and antimicrobials was examined by microdilution in broth and sterilized tap water. The effect of inhibitory and synergistic subinhibitory concentrations of common juniper EO and immortelle EO on biofilm of NTM on abiotic and biotic surfaces was investigated. The mechanism of action of common juniper EO has been studied by determining the degree of cell wall damage and morphological changes using a transmission electron microscope (TEM). Results: Common juniper and immortelle EO's proved to be most effective against NTM, while α-pinene tested alone showed poorer effect than overall EO. Common juniper EO have shown a number of synergistic effects, according to NTM, in combination with immortelle and white cedar EO and antimicrobials. The adhesion of NTM to polystyrene as well as biofilm formation were significantly inhibited (p < 0.05) with increasing temperature and increasing concentration of common juniper EO. Synergistic combinations of common juniper EO and antimicrobials have caused increased leakage of NTM cell macromolecules. TEM showed changes in the shape and structure of NTM after the activity of common juniper EO and synergistic effects in combination with immortelle EO. Conclusion: We have found a number of synergistic combinations of EO and antimicrobials that may have potential applications in new ways of preventing the adherence and formation of NTM biofilms not only in the water supply system, but also in artificial materials used in medicine and in biological systems. Synergies with the combined use of EO and antimicrobials allow the use of low effective concentrations with the sustained antimicrobial effect of the test substances with reduced toxicity. Common juniper EO cause changes in the cytoplasm and damage to the NTM cell wall, as evidenced by the observed increased leakage of cellular macromolecules and micromorphological changes observed by TEM

ANTIMICROBIAL EFFECT OF ESSENTIAL OILS ON NONTUBERCULOUS MYCOBACTERIA

Ciljevi istraživanja: Ispitati antimikrobni učinak eteričnih ulja (EU) odabranih mediteranskih biljaka (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis i Thuja occidentalis) i α-pinena na netuberkulozne mikobakterije (NTM) M. avium, M. intracellulare i M. gordonae te istražiti ulogu navedenih supstancija na biofilm NTM. Željeli smo utvrditi postojanje sinergističkih učinaka ispitivanih EU te kombinirane primjene EU i odabranih antimikrobnih lijekova i odrediti mehanizam djelovanja EU borovice na NTM. Metode: Uspostavom nove metode omogućeno je in vitro ispitivanje antimikrobnog učinka EU na NTM oponašajući uvjete vodoopskrbnih sustava. Određena je minimalna efektivna koncentracija (MEK) kao najmanja djelotvorna koncentracija EU u vodi. Metodom mikrodilucije u bujonu i steriliziranoj vodovodnoj vodi ispitana je osjetljivost NTM na odabrana EU i antimikrobne lijekove. Istražen je učinak inhibitornih i sinergističkih subinhibitornih koncentracija EU borovice i smilja na biofilm NTM na neživim površinama i površinama živih stanica. Mehanizam djelovanja EU borovice proučen je utvrđivanjem stupnja oštećenja stanične stijenke i morfoloških promijena primjenom transmisijskog elektronskog mikroskopa (TEM). Rezultati: EU borovice i smilja pokazala su se najučinkovitijima prema NTM, dok je α-pinen testiran samostalno pokazao slabiji učinak nego cjelovito EU. EU borovice iskazalo je u kombinaciji s EU smilja i tuje te antimikrobnim lijekovima niz sinergističkih učinaka prema NTM. Adhezija NTM na polistiren kao i formiranje biofilma, statistički značajno su inhibirani s porastom temperature i porastom koncentracije EU borovice. Sinergističke kombinacije EU borovice i antimikrobnih lijekova izazvale su povećano istjecanje staničnih makromolekula NTM. TEM uočene su promjene u obliku i strukturi NTM nakon djelovanja EU borovice i sinergističko djelovanje u kombinacijama s EU smilja. Zaključak: Pronašli smo niz sinergističkih kombinacija EU i antimikrobnih lijekova koje mogu imati potencijalnu primjenu u novim načinima sprječavanja adherencije i formiranja biofilma NTM ne samo u vodoopskrbnom sustavu, nego i na umjetnim materijalima koji se koriste u medicini te u biološkim sustavima. Sinergizam prilikom kombinirane primijene EU i antimikrobnih lijekova omogućuju primjenu niskih efektivnih koncentracija s održanim antimikrobnim učinkom ispitivanih supstancija s potencijalno smanjenom toksičnošću. EU borovice izaziva promjene u citoplazmi i oštećenje stanične stijenke NTM što je potvrđeno opaženim povećanim istjecanjem staničnih makromolekula i mikromorfološkim promjenama uočenim TEM.Research objectives: To investigate the antimicrobial effect of essential oils (EO) of selected Mediterranean plants (Juniperus communis, Helichrysum italicum, Lavandula hybrida, Salvia officinalis, Laurus nobilis and Thuja occidentalis) and α-pinene on non-tuberculous mycobacteria (NTM), M. avium, M. intracelullare and M. gordonae and investigate the role of these substances on NTM biofilm. We wanted to determine the synergistic effects of the tested EO and combinations of EO and selected antimicrobials. The aim was also to determine the mechanism of action of common juniper EO on NTM. Methods: The establishment of a new method enabled the in vitro testing of the EO's antimicrobial effect on NTM by mimicking the conditions of water supply systems. The Minimum Effective Concentration (MEC) was determined as the lowest effective EO concentration in water. The sensitivity of NTM to selected EO and antimicrobials was examined by microdilution in broth and sterilized tap water. The effect of inhibitory and synergistic subinhibitory concentrations of common juniper EO and immortelle EO on biofilm of NTM on abiotic and biotic surfaces was investigated. The mechanism of action of common juniper EO has been studied by determining the degree of cell wall damage and morphological changes using a transmission electron microscope (TEM). Results: Common juniper and immortelle EO's proved to be most effective against NTM, while α-pinene tested alone showed poorer effect than overall EO. Common juniper EO have shown a number of synergistic effects, according to NTM, in combination with immortelle and white cedar EO and antimicrobials. The adhesion of NTM to polystyrene as well as biofilm formation were significantly inhibited (p < 0.05) with increasing temperature and increasing concentration of common juniper EO. Synergistic combinations of common juniper EO and antimicrobials have caused increased leakage of NTM cell macromolecules. TEM showed changes in the shape and structure of NTM after the activity of common juniper EO and synergistic effects in combination with immortelle EO. Conclusion: We have found a number of synergistic combinations of EO and antimicrobials that may have potential applications in new ways of preventing the adherence and formation of NTM biofilms not only in the water supply system, but also in artificial materials used in medicine and in biological systems. Synergies with the combined use of EO and antimicrobials allow the use of low effective concentrations with the sustained antimicrobial effect of the test substances with reduced toxicity. Common juniper EO cause changes in the cytoplasm and damage to the NTM cell wall, as evidenced by the observed increased leakage of cellular macromolecules and micromorphological changes observed by TEM

Borrelia burgdorferi – emergent spirochete in Primorsko-goranska County

Cilj rada: Retrospektivno istražiti pojavnost lajmske borelioze (LB) u Primorsko-goranskoj županiji (PGŽ) u periodu od 1. siječnja 2018. do 31. prosinca 2019. Materijali i metode: Retrospektivna analiza rezultata serodijagnostičkih metoda pretrage 1843 uzorka krvnog seruma pacijenata s epidemiološkim podatkom o ugrizu krpelja. Dobiveni rezultati uspoređeni su s podatcima HZJZ-a o prijavljenim slučajevima LB-a na području Republike Hrvatske (RH) i PGŽ-a. Rezultati: Od 1050 uzoraka pretraženih tijekom 2018. godine na prisustvo specifičnih protutijela, 34,1 % imalo je pozitivna IgM protutijela na B. burgdorferi, dok su u uzorcima iz 2019. godine specifična protutijela detektirana u 26,1 % od 793 uzorka. Porast broja pozitivnih uzoraka uočen je u razdoblju od svibnja do rujna. U 2018. godini najveći je broj uzoraka s pozitivnim IgM protutijelima bio u lipnju (19,83 %), a 2019. godine u srpnju (18,35 %). Najmanji broj pozitivnih uzoraka u obje promatrane godine zabilježen je u siječnju i prosincu. U razdoblju od 2009. do 2019. godine HZJZ je prijavio u RH 510 ± 115,71 oboljelih godišnje. U 2019. godini broj prijavljenih slučajeva u PGŽ-u iznosio je 42,3 % od ukupnog broja slučajeva prijavljenih u RH. Zaključci: Velik broj oboljelih od LB-a u RH i PGŽ-u predstavlja sve veći javnozdravstveni problem. Ova zoonoza koja se prenosi krpeljima iz roda Ixodes pokazuje sezonski karakter, no zbog promjena klimatskih uvjeta, prvenstveno blažih zima, neophodna su istraživanja rasprostranjenosti krpelja u prirodi i preventivne mjere od infekcije.Objective: To investigate retrospectively the incidence of Lyme boreliosis (LB) in the Primorsko Goranska Counti (PGC) in the period from 1 January 2018 to 31 December 2019. Materials and Methods: A retrospective analysis of the results of serodiagnostic methods of examination of 1843 blood serum samples of patients with epidemiological data on tick bites. The obtained results were compared with the data of the Croatian Institute for Public Health on reported cases of LB in the territory of the Republic of Croatia (RC) and PGC. Results: Of the 1,050 blood serum samples screened during 2018 for the presence of specific antibodies 34.1% had positive IgM antibodies to B. burgdorferi while in 2019 samples, specific antibodies were detected in 26.1% of the 793 samples. An increase in the number of positive samples was observed in the period from May to September. In 2018, the largest number of samples with positive IgM antibodies was in June (19.83%), and in 2019 in July (18.35%). The lowest number of positive samples in both observed years was recorded in January and December. In the period from 2009 to 2019 Croatian Institute for Public Health reported in the RC number of 510 ± 115.71 patients per year. In 2019, the number of reported cases in PGC amounted to 42.3% of the total number of cases reported in RC. Conclusions: A large number of patients with LB in the RC, as well in PGC, is a growing public health problem. This zoonosis transmitted by ticks of the genus Ixodes shows a seasonal character, but due to changes in climatic conditions, primarily milder winters, it is necessary to research the distribution of ticks in nature and implement preventive measures against infection

Combined Application of Juniperus communis Essential Oil and Amikacin, Clarithromycin and Rifampicin against Mycobacterium avium and Mycobacterium intracellulare

The group of nontuberculous mycobacteria (NTM) includes about 200 mycobacteria that are widespread in the natural environment as free-living saprophytic bacteria, commensals or symbionts. NTM, also referred to as atypical mycobacteria, are mostly apathogenic; nowadays, they are increasingly important environmental opportunistic pathogens. This study continues the work of previous studies which investigated the individual and synergistic effect of different essential oils (EOs) on NTM. The aim was to investigate the effect of the interaction of the common juniper (Juniperus communis) EO and the antimicrobials, amikacin, clarithromycin and rifampicin, against Mycobacterium avium and M. intracellulare using the checkerboard synergy method in an enriched Middlebrook 7H9 broth. Morphological changes of treated NTM cells were observed with a transmission electron microscope. The most synergistic combinations were found at subinhibitory concentrations of the common juniper EO and rifampicin against both tested NTM and this EO and clarithromycin against M. avium. A slightly smaller number of synergistic effects on both NTM were found using a combination of this EO and amikacin. Combinations of clarithromycin and the common juniper EO showed no synergism against M. intracellulare. The exposure of both NTM to synergistic combinations of this EO and antimicrobials caused significant morphological changes in mycobacterial cells. Synergism with the combined use of EOs and antimicrobials allows the use of low effective concentrations via the sustained antimicrobial effect of the tested substances, but with potentially reduced toxicity

Eterična ulja borovice (Juniperus communis) i smilja (Helichrysum italicum) sinergijski inhibiraju adheziju netuberkuloznih mikobakterija na Acanthamoeba castellanii

Acanthamoeba is an opportunistic protozoon, widespread in the aquatic environment, where it can be in endosymbiosis with over 30 pathogenic bacteria, including nontuberculous mycobacteria (NTM). Protozoa play a crucial role in mycobacterial pathogenesis and serve as a reservoir of infection. Since the first step in bacteria making contact with amoebae is adhesion, we were interested in investigating whether essential oils (EOs) can affect it. To that end we investigated the effects of juniper (Juniperus communis) and immortelle (Helichrysum italicum) EOs against Mycobacterium avium, M. intracellulare, and M. gordonae in tap water and against their adhesion to Acanthamoeba castellanii by combining them in synergistic EO concentrations. M. avium and M. intracellulare adhered to A. castellanii to a greater extent than M. gordonae. The adhesion of all NTMs was prevented by the subinhibitory concentrations of EOs. When comparing the effect of synergistic combinations of EOs and the effect of a single concentration from a combination, a higher percentage of adhesion inhibition in all synergistic combinations observed, except against M. gordonae. Neither oil was cytotoxic to A. castellanii. Our findings suggest that the EOs or their components weaken the contact of environmental NTMs and free-living amoebae and indirectly diminish their pathogenic potential, which could be of value in developing strategies for maintenance of water supply systems.Akantameba je oportunistička protozoa, široko rasprostranjena u vodenom okružju, gdje može kao endosimbionte sadržavati više od 30 različitih patogenih bakterija, uključujući netuberkulozne mikobakterije (NTM). Protozoe kao rezervoar infekcije imaju ključnu ulogu u mikobakterijskoj patogenezi. Sposobnost mikobakterija da se vežu, ulaze I umnožavaju se unutar protozoa vjerojatno im je omogućila da postanu intracelularni patogeni. Budući da je adhezija prvi korak u uspostavi kontakta bakterija s amebama, zanimalo nas je istražiti mogu li prirodni spojevi poput eteričnih ulja utjecati na taj kontakt. Cilj ove studije bio je istražiti učinak eteričnih ulja borovice (Juniperus communis) i smilja (Helichrysum italicum) prema NTM-ovima (M. avium, M. intracellulare i M. gordonae) u pitkoj vodi. Također, ispitana je mogućnost inhibicije adhezije tih NTM-ova na Acanthamoeba castellanii (A. castellanii) primjenom sinergijskih koncentracija eteričnih ulja. M. avium i M. intracellulare, u odnosu na M. gordonae, u većoj su mjeri adherirali na A. castellanii. Adhezija svih NTM-ova inhibirana je subinhibicijskim koncentracijama eteričnih ulja borovice i smilja. Kad se usporedi učinak sinergijskih kombinacija i učinka pojedine koncentracije iz sinergije, uočen je veći postotak inhibicije adhezije kod primjene svih sinergijskih kombinacija, osim jedne prema M. gordonae. Oba eterična ulja u ispitivanim koncentracijama nisu imala citotoksično djelovanje na stanice A. castellanii. Na taj su način primijenjene subinhibicijske koncentracije eteričnih ulja sa smanjenim toksičnim učinkom, uz zadržan antimikrobni i antiadhezivni učinak. Primjena eteričnih ulja ili njihovih komponenata u umjetnom vodenom okolišu mogla bi oslabiti kontakt NTM-ova i slobodno živućih ameba i posredno dovesti do smanjenja patogenoga potencijala okolišnih NTM-ova

Prevalence of occult hepatitis B virus infection and characterisation of hepatitis B surface antigen mutants among adults in western Croatia

Introduction and Objectives: Occult hepatitis B virus (HBV) infection (OBI) is characterised by low levels of hepatitis B virus (HBV) DNA in the blood/liver of patients with negative hepatitis B surface antigen (HBsAg). This study aimed to determine the OBI prevalence and virological characteristics (viral genotypes and HBsAg mutants) in patients with an "anti-HBc only" serological profile. Materials and Methods: A total of 24 900 serum samples were routinely screened for hepatitis B markers over a five-year period. All anti-HBc-positive/HBsAg-negative/anti-HBs-negative sera were selected and analysed for the presence of HBV DNA. Mutational analyses of the HBs gene and polymerase gene sequences were performed. Results: 1749 (7.02%) sera were anti-HBc positive, and 113 (0.45%) sera had an "anti-HBc only" serological profile (HBsAg/anti-HBs negative). HBV DNA was detected in 12/113 (10.61%) "anti-HBc only" positive sera, representing 0.048% of all routinely tested samples. Due to extremely low viremia, HBV genome was successfully sequenced in only two sera where subgenotype D3 was confirmed. Mutational analyses of the S gene revealed multiple missense mutations. In addition to the M133I, Y134F, and G145R mutations, already associated with diagnostic escape, we also found nine novel OBI-related S-gene mutations - S136Y, F158L, K160N, E164G, S167L, A168V, L175S, S210I and F212C. Conclusions: We detected multiple known and novel S gene mutations in 2/12 (16.6%) OBI cases, nevertheless, further studies are required to determine their role in the pathogenesis of OBI. Understanding the frequencies of clinically relevant HBV mutations may contribute to improvement of diagnostic protocols