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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.

Formation of biofilms allows bacterial cells to survive in adverse environments. Nontuberculous mycobacteria are ubiquitous in aqueous environments, where they adhere to surfaces and create a biofilm. This has led to the emergence of healthcare-associated infections and the use of biomaterials in medicine. Essential oils (EO) are substances of natural origin whose effect on microorganisms has been the subject of numerous studies. Here, we investigated the effect of Juniperus communis EO on nontuberculous mycobacteria and their early and mature biofilm formation in sterilised tap water. The combination of Juniperus communis EO and increasing ambient temperature showed a synergistic effect on the reduction of biofilm formation of Mycobacterium avium and Mycobacterium intracellulare on a polystyrene surface. A significant antibiofilm effect of Juniperus communis EO was also found at subinhibitory concentrations, suggesting a potential role for it as an alternative disinfectant of natural water.

Objective: The present study evaluated the possible synergistic antimycobacterial interactions of Juniperus communis and Helichrysum 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, minimum bactericidal concentration (MBC) and minimal effective concentration (MEC) were 1.6 mg ml -1 for J. communis EO and 3.2 mg ml -1 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 EOs 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.

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.

The aim of the study was to investigate the molecular epidemiology of community- associated MRSA in Primorsko-Goranska County of Croatia during a six-year period(2001-2007). In period from 2001 and 2007, 46 MRSA isolates were collected in Rijeka, strains were subjected to susceptibility testing according to CLSI guidelines, mecA gene detection and SCCmec typing as well as detection of PVL. Strains were typed by Pulse Field Gel Electrophoresis (PFGE) and spa typing. All isolates were susceptible to vancomycin, linezolid, mupirocin, nitrofurantoin, only one strain was resistant to fusidic acid and co-trimoxazole. Results of SCCmec typing showed the presence of SCCmec type IV in 26 MRSA strains, SCCmec type V in three strains, and 13 strains comprised SCCmec I. SCCmec type II and III were not observed. Four MRSA strains were non-typeable by applied SCCmec typing methods. PVL was detected in 4 strains, two SCCmec IV and two SCCmec V. PFGE analysis, grouped MRSA strains into six similarity groups and 18 singletons. Dominating spa types in this collection of strains were t015, with 15 strains, followed by t041(N=7), t051,(N=2 ), t2850(N=2), t008(N=2)and single isolates t441, t002, t448, t018, t019, t355, t390, t026, t449, t148. We also detected two new spa types, t3510 and t3509, respectively. This is the first report on SCCmec type V in Croatia, and, to our knowledge, first report of PVL-positive mehicillin-resistant Staphylococcus aureus SCCmec type V and t441(ST59-MRSA-V) in this part of Europe.

The aim of the study was to investigate campylobacteriosis incidence in the County Primorsko-goranska (Croatia) between 2003 and 2007 and to find out possible connection with environmental factors (the average monthly temperature and total monthly precipitation). The data (number of stool samples examined, age and sex distribution of patients, monthly distribution of isolates and distribution of isolates according to the species) from the Laboratory for Diagnostics of Enteric Infections of the Teaching Institute of Public Health of the County Primorsko-goranska (Croatia) were analyzed retrospectively. During the observed period 30,164 stool samples were examined for Campylobacter spp. Campylobacters were identified in 1,242 (4.12%) samples. The overall annual campylobacter incidence rate was 81.3 +/- 21.9/100,000 population. Campylobacter jejuni was found in 1,093 (88%) and C. coli in 149 (12%) patients. Our findings showed age distribution of patients typical for developed countries. The patients were mostly children under 5 years (484.4 +/- 129.1/ 100,000, p < 0.001) and between 5 and 9 years of age (226.5 +/- 60.5/100,000, p < 0.05). Male consistently experienced higher rates, but the difference between genders was significant in the age groups from birth till late twentieth (p < 0.001). Campylobacter rates were significantly associated with monthly average temperatures (p < 0.05), but not with precipitation. Further investigations into the incidence of campylobacteriosis on the national level are necessary. The causes of the noticed monthly distribution, sources of infection and connection with the routes of transmission in humans need to be elucidated as well.

The purpose of this study was to determine the presence of individual microorganisms and the most frequent microbial combinations in the biofilm of the indwelling Provox2 voice prosthesis in situ. Furthermore, we wanted to evaluate the possible influence of biofilm composition on the mean and median lifetime of these voice prostheses. Over a 5-year period, implantation of a Provox2 voice prosthesis was performed in 85 patients, or 90% of the overall number of patients who underwent total laryngectomy. In total 100 implanted voice prostheses, at least one of every patient, were microbiologically processed immediately after being replaced. Out of the total of 292 isolates, 67% were bacteria and the remaining 33% were yeasts. The most frequently found yeast species on voice prostheses biofilms was C. albicans, followed by C. krusei and C. tropicalis. The most frequently isolated bacteria included Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Streptococcus agalactiae. Simultaneous presence of bacteria and fungi was established in 83% of the processed voice prostheses; in 16% of samples the biofilm contained only one or more bacterial species. The mean time of implantation was 238 days and the median lifetime of the device was 180 days. Dividing the prostheses in four groups according to the composition of biofilm revealed that the device lifetimes varied significantly between groups. The longest lifetime of voice prostheses was associated with the presence of single fungal isolate in combination with bacteria. There is a significant correlation between biofilm composition and the device life time.

The aim of this study was to report the prevalence of uropathogens and the frequency of extended spectrum beta-lactamases (ESBL) producing strains isolated from urine of outpatients in Primorsko-Goranska County in Croatia. We have retrospectively analyzed the results of 44, 321 urine cultures from January 01, 2008 till June 30, 2009. The study showed that ESBL production was confirmed in 189 (1.8%) of the total of 10,757 isolates. Rates of ESBL-producing isolates were 19%, 0,6%, and 5.2% for Klebisella pneumoniae, Escherichia coli and Proteus mirabillis, respectively. Geographic variations in pathogen occurrence and susceptibility profiles require continuous monitoring to provide information to guide the empiric therapeutic options.