Interaction between Acanthamoeba and Staphylococcus.

Free-living amoebae of the genus Acanthamoeba are infected by various bacteria in nature, and thus bacteria can protect themselves from adverse environmental conditions. Contrary to this ameba-bacteria relationship whether Acanthamoeba has antibacterial effects on bacteria is the different aspect of the relationship between these microorganisms. In this study, we investigate various Acanthamoeba strains have antibacterial effects on various Staphylococcus strains. Three environmental Acanthamoeba strains, isolated from various aquatic environments in Turkey, and Acanthamoeba castellanii ATCC 50373 standard strains were used in the study. The antistaphylococcal effect of cell-free supernatant (CFS) obtained from these amoebae against 12 different Staphylococcus bacteria was investigated by colony counting method. In addition, the pathogenicity of the tested Acanthamoeba strains was determined using osmotolerance and thermotolerance tests. CFSs obtained from Acanthamoeba were found to have varying degrees of antistaphylococcal effects on various Staphylococcus strains (0%-100%). It was determined that the CFS of the standard Acanthamoeba strain showed 100% inhibitory effect against one clinical methicillin-resistant Staphylococcus aureus strain (M2). Also, CFS of Ugöl strain showed 99.97% inhibitory effect against one clinical methicillin-sensitive Staphylococcus epidermidis strain (L3). It was determined that all Acanthamoeba isolates had no pathogenic potential. According to the results, it has been observed that Acanthamoeba produces antibacterial substance(s) against Staphylococcus bacteria and that the ameba-bacteria relationship may also result in the detriment of the bacteria. Furthermore, the current study indicates that new and natural antimicrobial agents from Acanthamoeba can be used as an alternative to infections caused by Staphylococcus.

[Investigation of the Inhibition Effect of Acanthamoeba Cell-Free Supernatants Against Pseudomonas aeruginosa]. / Acanthamoeba Hücresiz Sivilarinin Pseudomonas aeruginosa Karsisinda Inhibe Edici Etkisinin Arastirilmasi.

Free-living amoebae belonging to the genus Acanthamoeba are microorganisms that live in air, soil and aquatic environments. In humans, they cause infections such as amoebic keratitis, graulamotous amoebic encephalitis that are difficult to treat and can be fatal. In addition, it is known that they contribute to the replication of bacteria and increase their pathogenicity by being a host for various bacteria. However, information on its inhibitory properties against bacteria and its production of antimicrobial agents is very limited. In this context, in this study, it was aimed to investigate whether cell-free supernatants of Acanthamoeba strains have antibacterial effects against Pseudomonas aeruginosa isolates. Four different Acanthamoeba strains (A10, A13, A14, U.GÖL) isolated from aquatic environments in our country were selected and used in the study, P.aeruginosa isolates (PA2, PA3, PA4, PA5) were selected from clinical strains belonging to patients in our country. Acanthamoeba castellanii ATCC 50373 and P.aeruginosa ATCC 27853 were used as standard strains. P.aeruginosa isolates were grown on nutrient agar at 37 °C and Acanthamoeba strains were grown on E.coli spread non-nutrient agar at 30 °C under aerobic conditions. Pepton yeast extract glucose (PYG) medium supplemented with penicillin and streptomycin was used to obtain axenic cultures of Acanthamoeba strains. After the centrifugation of axenic cultures at 3000 rpm for five minutes, Acanthamoeba-cell-free supernatants were obtained by filtering the supernatant part through a sterile filter with a pore diameter of 0.22 µm. The antibacterial activities of these supernatants against P.aeruginosa isolates were determined using the colony counting method. Analysis of each Acanthamoeba-cell-free supernatants was performed according to the GC-MS method. Acanthamoeba-cell-free supernatants were found to have varying degrees of inhibitory effects (3.9-91.5%) against tested P.aeruginosa isolates. It was determined that the cell-free supernatant of A.castellanii ATCC 50373 strain showed the highest antibacterial effect (91.5%) against PA5 isolate. A14 strain showed similar inhibitory effects (89.4%) against the same Pseudomonas isolate. The average inhibitory effect of most of the Acanthamoeba strains of our country was found to be higher than that of the reference strain A.castellanii ATCC 50492. It is thought that the compounds responsible for the anti-Pseudomonas activity of the tested Acanthamoeba strains may be fructose, phosphoric acid, galactose, N-Acetylphenylalanine and glucopyranose determined as major compounds. This is the first study showing the anti-Pseudomonas activity of microorganisms of the genus Acanthamoeba living in the waters of our country. Acanthamoeba, which is widely found in nature, appears to be a good source for new antimicrobial agents.

[Inhibition Effect of Cell-Free Supernatants of Environmental Acanthamoeba Strains on the Viability of Metastatic Cell-Lines]. / Çevresel Acanthamoeba Suslarinin Hücresiz Sivilarinin Metastatik Hücre Hatlarinin Canliligi Üzerindeki Inhibisyon Etkisi.

It is known that some of the therapeutic agents against cancer cells are isolated from natural sources such as plants and animals. However, due to increasing drug resistance, studies on the discovery of new sources are needed. In this study, it was aimed to investigate the inhibition effects of four native Acanthamoeba strains on different cancer cell lines (MDA-MB-231, PC3, MAT-LyLu). 3T3 cells were used as normal cell line. All strains were recultured by using non-nutrient agar spread by heat-inactivated Escherichia coli ATCC 25922. A.castellanii ATCC 50373 was used as the standard strain. Molecular identification of the native Acanthamoeba isolates was done by polymerase chain reaction (PCR) and DNA sequence analysis using specific primer pairs (P-FLA-F, P-FLA-R, JDP-F, JDP-R). Axenic cultures of all strains were obtained in 25 cm2 tissue culture flasks and in peptone yeast extract glucose (PYG) medium. In order to investigate the effect of cell-free supernatants obtained from axenic cultures on cancer cell lines and 3T3 cell viability, MTT method was applied using different concentrations of cell-free supernatants (1%, 2%, 5%, 10%, 15%). It was determined that the viability of 3T3 cells was not affected by any Acanthamoeba cell-free supernatants (p≤ 0.05). All of the samples tested were found to have a significant inhibitory effect (p<0.05) on the viability of PC3 and MAT-LyLu cells (human and rat prostate cancer cell line). However, none of the samples had an inhibitory effect on the viability of MDA-MB-231 (breast cancer cell-line). Two native Acanthamoeba cell-free supernatants showed higher inhibitory potency (28% and 21.9%) at 2% concentration against PC3 cells compared to the reference strain (16%). Similarly, the same Acanthamoeba samples were also shown to have a better inhibition potential on the viability of MAT-LyLu cells than the reference strain. It was found that the inhibitory potential of Acanthamoeba cell-free supernatants may not be related to proteins and proteases. The results obtained from this study showed that Acanthamoeba species living in the aquatic environment isolated from our country have a potential inhibitory effect against the tested cancer cell lines. In addition to plants and animals, Acanthamoeba cell-free supernatants can also be a source for natural therapeutic substances that act against cancer cells. However, it is necessary to carry out new studies using more strains in order to detect strains with higher inhibitory effects.

[Investigation of the Incidence of Legionella and Free-Living Amoebae in Swimming Pool Waters and Biofilm Specimens in Istanbul by Different Methods]. / Istanbul'daki Yüzme Havuzu Sulari ve Biyofilm Örneklerinde Legionella ve Serbest Yasayan Amiplerin Görülme Sikliginin Farkli Yöntemlerle Arastirilmasi.

Legionella bacteria living in free form or in biofilm and free-living amoebae (FLA) can infect humans through swimming pools and can cause various diseases. FLA may also threaten the health of swimmers because they are capable of being hosts for Legionella and some other bacteria. The aim of this study was to investigate the presence of total aerobic heterotrophic bacteria (TAHB), FLA and Legionella bacteria in swimming pool waters and biofilm samples in Istanbul by using culture and FISH methods. Water plate count agar (wPCA), buffered charcoal yeast extract (BCYE) agar supplemented with glycinevancomycin-polymyxin-cycloheximide (GVPC) and Escherichia coli cultivated non-nutrient agar (NNA) were used for the culture of TAHB, Legionella and FLA. For the FISH method analysis , Leg 705 and Leg PNE1 probes labeled with fluorescent dye for Legionella and ACANTHA probe for Acanthamoeba genus FLA were used. Legionella pneumophila serogroup 1 ATCC 33152, L.pneumophila serogroup 3 ATCC 33155 and Acanthamoeba castellani ATCC 50373 were used as positive controls. TAHB were grown in 92% and 84% of water and biofilm samples. Although Legionella bacteria could not be grown in any of the water samples, it was detected in 6 (24%) water samples by FISH method. Although these bacteria could be grown in 1 (4%) of biofilm samples, 7 (28%) were detected by FISH method. FLA were found to be 16% by culture in water samples and 28% by FISH analysis. These amoebae were detected 8% and 20% in biofilm samples by culture and FISH method, respectively. It was determined that one of the isolates of FLA had thermotolerant activity (potentially pathogenic). L.pneumophila serogroup 1 was detected in one water sample and in four biofilm samples. According to the culture method, TAHB and FLA were found to be more common in water samples than in biofilm samples and Legionella bacteria were more common in biofilm samples than in water samples (p≤ 0.05). In the detection of Legionella bacteria, the superiority of FISH method compared to culture method was found to be statistically significant (p≤ 0.05). In this study, it was found that the number of TAHB in the controlled swimming pools was within the limits determined by the Ministry of Health (≤ 200 cfu/ml). It will be appropriate to examine both water and biofilm samples for the investigation of TAHB, FLA and Legionella. It may be appropriate to use both culture and FISH methods to detect the presence of FLA in water and biofilm samples. This study is the first study to investigate the presence of Legionella and FLA in swimming pools in Istanbul, and further studies are needed to examine more pool water and biofilm samples. With the data obtained, the health principles and controls of swimming pools will be re-considered and will be contributed to public health.

In vitro activities of antimicrobial peptides and ceragenins against Legionella pneumophila.

Legionella pneumophila is a waterborne intracellular pathogenic bacterium, the most frequent cause of human legionellosis and a relatively common cause of community-acquired and nosocomial pneumonia. Some legionellosis outbreaks are related to the presence of biofilms, which provide a reservoir for L. pneumophila strains. We investigated the in vitro activities of antibiotics; erythromycin and doxycycline, antimicrobial peptides AMPs; melittin, LL-37 and CAMA (cecropin A (1-7)-Melittin A (2-9) and ceragenins; CSA-8, CSA-13, CSA-44, CSA-131 and CSA-138 against L. pneumophila. Isolation of Legionella strains was conducted according to ISO 1998. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum biofilm eradication concentrations (MBECs) were determined using microbroth dilution techniques. MIC ranges for melittin, LL-37, and CAMA were 0.25-1, 1-4, and 2-8 µg ml-1, respectively. MIC ranges for CSA-8, 13, 44, 131, and 138 were 0.5-2, 0.5-1, 1-4, 0.5-2, and 1-2 µg ml-1, respectively, and MBEC values for the ceragenins were 10-160 µg ml-1. These results demonstrate that AMPs and ceragenins display broad-spectrum, in vitro activity against L. pneumophila. In particular, CSA-8, CSA-13 and melittin gave the lowest MICs and MBCs. We also observed that ceragenins are active against established L. pneumophila biofilms.

Growth ability of Gram negative bacteria in free-living amoebae.

When bacteria and free-living amoebae (FLAs) live both in natural waters and man-made aquatic systems, they constantly interact with each other. Some bacteria can survive and grow within FLAs. Therefore, it has recently been thought that FLAs play an important role in spreading pathogenic bacteria in aquatic systems. In this study we investigated the intracellular growing ability of 7 different Gram-negative bacteria (Pseudomonas fluorescens, Pseudomonas putida, Pasteurella pneumotropica, Aeromonas salmonicida, Legionella pneumophila serogroup 1, L. pneumophila serogroup 3, L. pneumophila serogroup 6) in four different FLA isolates (A1-A4). Among these, four bacterial isolates (P. fluorescens, P.putida, P.pneumotropica, A.salmonicida) and two free-living amoebae isolates (A3, A4) were isolated from the tap water in our city (Istanbul). It was found that 4 different Gram-negative bacteria could grow in A1, 2 different Gram-negative bacteria could grow in A2, 4 different Gram-negative bacteria could grow in A3, 1 Gram-negative bacterium could grow in A4. In conclusion, we think that this ability of growth could vary according to the characteristics of both bacteria and FLA isolates. Also, other factors such as environmental temperature, bacterial concentration, and extended incubation period may play a role in these interactions. This situation can be clarified with future studies.

Amoebicidal efficacy of a novel multi-purpose disinfecting solution: first findings.

Free-living amoebae of the genus Acanthamoeba can cause a sight-threatening corneal infection, Acanthamoeba keratitis, mostly in contact lens wearers. The use of ineffective contact lens disinfecting solutions is one of the most important risk factors for this infection. This study concerns a new multi-purpose contact lens disinfecting solution, OPTI-FREE® PureMoist®, tested for its efficacy against Acanthamoeba trophozoites and cysts by using the most probable number technique for amoebic enumeration. Acanthamoeba castellanii ATCC 50373 and an environmental strain of Acanthamoeba genotype T4 isolated from tap water in Istanbul were used during the experiments. OPTI-FREE® PureMoist® achieved total kill (more than a 3-log reduction) of trophozoites of both strains before the manufacturer-recommended disinfection time (6h). In contrast, this solution had limited cysticidal activity against the ATCC strain but more against the environmental strain, with log reductions of 0.75 and 2.20, respectively, after 6h of exposure.

Microbial contamination of contact lens storage cases and domestic tap water of contact lens wearers.

Contact lenses have been widely used as an alternative to spectacles both in developed and developing countries. However, under certain circumstances, adverse responses can occur during contact lens wear and several microorganisms--including bacteria, fungi, and free living amoebae--can cause several eye infections in wearers. Extended wear of contact lenses is the major risk factor of eye infections such as microbial keratitis, besides contaminated contact lens storage case, contaminated lens care solutions, and inaccurate contact lens handling. In this study, we collected contact lens storage case and domestic tap water samples from 50 asymptomatic contact lens wearers. We determined that total aerobic mesophilic bacteria were isolated in 45 (90 %), Gram negative rod bacteria were isolated in 20 (40 %), Pseudomonas spp. were isolated in 2 (4 %) and fungi were isolated in 18 (36 %) out of 50 contact lens storage cases. Free living amoebae were not detected in investigated contact lens storage cases. At the same time, out of 50, total aerobic mesophilic bacteria were isolated in 34 (68 %), fungi were isolated in 15 (30 %) and free living amoebae were isolated in 15 (30 %) domestic tap water samples. No Gram-negative rod bacteria and Pseudomonas spp. were detected in investigated water samples. Two contact lens case samples and two tap water samples were excluded from the analysis for Pseudomonas spp. for technical reasons. According to our findings, inadequate contact lens maintenance during lens wear may result in the contamination of contact lens storage cases. This situation can lead to severe eye infections in contact lens wearers over time.

Postantibiotic effect of various antibiotics on Legionella pneumophila strains isolated from water systems.

The postantibiotic effects (PAE) of azithromycin, clarithromycin, ciprofloxacin, and levofloxacin were investigated against Legionella pneumophila (L. pneumophila) strains isolated from several hot water systems of different buildings in Istanbul. Each strain in logarithmic phase of growth was exposed to concentrations of antibiotics equal to minimum inhibitory concentration (MIC) and 4× MIC for 1 h. Recovery periods of test cultures were evaluated after centrifugation using the viable counting method. The mean values of PAEs for the strains of L. pneumophila, azithromycin at a concentration equal to and 4 times of MIC values were found 1.75 ± 0.28 h and 4.06 ± 0.44 h, for clarithromycin 2.98 ± 0.70 h and 4.18 ± 0.95 h, for ciprofloxacin 2.97 ± 0.63 h and 4.70 ± 0.63 h, for levofloxacin 2.05 ± 0.33 h and 3.78 ± 0.46 h, respectively. All of the antibiotics showed increased PAE values in a concentration-dependent manner. The findings of our study may play useful role in selecting the appropriate timing of doses during therapy with antimicrobials to treat patients infected with L. pneumophila.

Profiling of environmental Legionella pneumophila strains by randomly amplified polymorphic DNA method isolated from geographically nearby buildings.

Legionella pneumophila (L. pneumophila) which is also known as etiologic agent Legionnaires Disease lives in natural water and man made water systems. These bacteria belonging to Legionellaceae family are divided 15 serogroups. Phenotypical methods used for the identification of Legionella isolates are not very discriminatory. In this study we investigated genotypic features of eight L. pneumophila serogroup 1 and 18 L. pneumophila serogroup 2-14 strains isolated from different buildings in Istanbul by randomly amplified polymorphic DNA (RAPD) method. Eight L. pneumophila serogroup 1 strains (37.5%) were similar RAPD profile and they were isolated from buildings located in a short distance (about 500 m). Four L. pneumophila serogroup 2-14 strains (22%) were identical genotypically. Three of these strains were isolated from buildings located in a short distance.

Microbial contamination of dental unit waterlines in Istanbul, Turkey.

The water used in dental unit waterlines (DUWLs) acts as a coolant for the high-speed equipment and as an irrigant during dental treatments. There are kind of water tanks. DUWLs provide a favorable environment for microbial biofilm and multiplation primarily due to the high surface in the tubing and the character of fluid dynamics in narrow, smooth-walled waterlines. Biofilms can harbour opportunist pathogens such as Legionella sp., Pseudomonas sp. Several studies have shown that DUWLs have high levels of microbial contamination. Presence of high level of microbial contamination is an important problem for dentists and dental patients who are immunocompromised. We collected water samples from DUWLs of 20 private dental offices. We have determined that only 2 (3.4%) out of 59 dental unit water samples were found to meet the standard (<200 CFU.ml(-1)) for DUWLs water quality by American Dental Association (ADA). Of the 59 water samples examined, 14 (24%) were positive for Pseudomonas sp. and 18 (30.5%) were positive for fungi. The most common 14 bacterial strains and seven fungi were isolated. Of bacterial strains, 57.1% were identified: Majority of the bacterial species isolated from our samples was identified as Pseudomonas fluorescens, Pasteurella haemolytica, Photobacterium damsela, Ochrobacter anthropi, Moraxella sp., Aspergillus flavus, Penicillium expansum. Legionella sp. were not detected in all water samples.

Isolation and identification of enterococci from seawater samples: assessment of their resistance to antibiotics and heavy metals.

A hundred Enterococcus strains were isolated from seawater samples collected from coastal areas of Istanbul. Isolates were identified to the species level using standard biochemical tests specified by Facklam and Collins. The species distribution was as follows Enterococcus faecalis (96%), Enterococcus gallinarum (3%) and Enterococcus solitarius (1%). The resistance of bacteria to both heavy metals (zinc [Zn], iron [Fe], cadmium [Cd], chrome [Cr], cobalt [Co]) and antibiotics (ampicillin 10 microg [AP], penicillin G 10 Units [PG], gentamycin 10 microg [GM], streptomycin 10 microg [S], chloramphenicol 10 microg [C], erythromycin 15 microg [E], kanamycin 30 microg [K], amikacin 30 microg [AK], nalidixic acid 30 microg [NA], and vancomycin 30 microg [VA]) was evaluated. None of the strains was resistant to VA. It was found that among the 100 isolates, those that exhibit resistance to antibiotics, particularly NA, S and K, were also resistant all the heavy metals tested. To our knowledge this is the first report focusing on determination of resistance of environmental enterococci found in Istanbul against heavy metals and antibiotics. Thus, combined expressions of antibiotic and heavy metal resistance may help to reinforce ecological and epidemiological studies and to determine the role of these strains in antibiotic and heavy metal resistance dissemination.