Soil transmitted helminths and schistosoma mansoni infections among school children in zarima town, northwest Ethiopia

Abebe Alemu
Asmamaw Atnafu
Zelalem Addis
Yitayal Shiferaw
Takele Teklu
Biniam Mathewos
Wubet Birhan
Simon Gebretsadik
Baye Gelaw
Soil transmitted helminths and schistosoma mansoni infections among school children in zarima town, northwest Ethiopia
R E S E A R C H A R T I C L E Open Access
Background: In Ethiopia, because of low quality drinking water supply and latrine coverage, helminths infections are the second most predominant causes of outpatient morbidity. Indeed, there is a scarcity of information on the prevalence of soil transmitted helminths and Schistosomiasis in Ethiopia, special in study area. Therefore, the aim of this study was to determine the prevalence and associated risk factors of soil transmitted helminths and intestinal Schistosomiasis.Methods: Cross-sectional study was conducted among 319 school children of Zarima town from April 1 to May 25, 2009. A pre-tested structured questionnaire was used to collect socio-demographic data and possible risk factors exposure. Early morning stool samples were collected and a Kato Katz semi concentration technique was used to examine and count parasitic load by compound light microscope. Data entry and analysis was done using SPSS-15 version and p-value < 0.05 considered statistically significant. Results: Out of 319 study subjects, 263 (82.4%) of the study participants infected with one or more parasites. From soil transmitted helminths, Ascaris lumbricoides was the predominant isolate (22%) followed by Hookworms (19%) and Trichuris trichiura (2.5%). Schistosoma mansoni was also isolated in 37.9% of the study participants. Hookworm and S. mansoni infections showed statistically significant associations with shoe wearing and swimming habit of school children, respectively.Conclusion: Prevalence of soil transmitted helminths (STH) and S.mansoni was high and the diseases were still major health problem in the study area which alerts public health intervention as soon as possible.

Background

Soil transmitted helminthic infections and Schistosomiasis are among the widely spread chronic infections in the world. Globally 2 billion individuals are infected with helminths, out of these majorities live in resource-poor settings [1,2]. World health organization (WHO) estimated the common STHIs in world as: 250 million cases for Ascariasis, 151 million cases of hookworm diseases, 100 million cases of Strongyloidiasis and 45.5 million cases of Trichuriasis [3]. Schistosomiasis is also remains one of the most prevalent parasitic diseases in the world. It is endemic in 76 countries and continues to be public health concern in developing countries. Approximately 80% of the 200 million people infected world-wide live in sub-Saharan Africa where Schistosoma mansoni and Schistosoma haematobium are widespread [4,5].

The occurrence of helminthic infections is associated with socioeconomic, environmental and other factors like, ignorance of simple health promoting factors and overcrowding, limited access to clean water, tropical climate and low altitude [2]. School age children are one of the groups at high-risk for intestinal parasitic infections. Factors like poor developments of hygienic habits, immune system and over-crowding contributes for infection [6]. The adverse effects of intestinal parasites among children are diverse and alarming. Intestinal parasitic infections have detrimental effects on the survival, appetite, growth and physical fatnesses, school attendance and cognitive performance of school age children [7][8][9][10][11].

Ethiopia has one of the lowest quality drinking water supply and latrine coverage in the world [12]. Because of this and other reasons intestinal parasites have been widespread in Ethiopia and parasitic helminthic infections are the second most predominant causes of outpatient morbidity in the country. Many reports illustrated that Ascariasis is the most prevalent intestinal parasitic infection in different communities usually occurring together with trichuriasis. Hookworm infection, Strongyloidiasis and Enterobiasis are also public health problem though the magnitude is lesser compared to Ascariasis [13]. Also schistosomiasis is common in northern region as compared to south and south west regions of Ethiopia [14].

Intervention against STH and Schistosoma infections are based on regular anti-helminthic treatment, improved water supply, sanitation and health education [15][16][17]. STH infections have not been targeted for control in Ethiopia [18], though mass de-worming as a component of the Enhanced Outreach Strategy (EOS) targeting under five children started in 2004 [19]. Low-cost, high-coverage delivery of anti-helminthic treatment has been achieved in some settings [20] but improving sanitation is more complex. In Ethiopia, for example, levels of access to improved sanitation in rural areas are very low (5.4%) making evaluation of other components of intervention important [21].

Prevalence of intestinal helminths and other intestinal parasites have been studied in different of tropics and subtropics including Ethiopia [22][23][24][25][26][27][28][29][30][31][32]. Although many studies previously conducted in Ethiopia, gave due attention to distribution of different intestinal parasites on different altitudes in different community groups such as preschool children, school children and other groups confined to camps and refugees, the prevalence of STH and schistosomiasis was not well addressed indifferent parts of Ethiopia including our study area. Therefore, the aim of this study was to determine the prevalence and associated risk factors of soil transmitted helminths and Schistosoma mansoni infection among school children in Zarima town, North Gondar, North West Ethiopia. As result this study envisages that it might strengthen the information so far for scaling up and to design effective communication strategy to combat STH and S. mansoni in the study area.

Methods

Study area

Cross-sectional study was conducted from April 1 to May 25, 2009 among elementary school children of Zarima town which is one of small town in "Adarkay" woreda in North Gondar. It is 140 km far from Gondar town. The town has approximately a total population of about 20, 000 [33]. The area is predominately rural and most residents live in villages as agriculturists which is mixed agriculture; growing maize, teff (Eragrostos teff) and wheat. The altitude of the town range from 1000-2400 meter above see level and the topography shows hills and plain land with springs, streams and rivers which are often source of water for domestic and other uses for the communities in the town and around the town. These predispose the children to water borne diseases during swimming, washing, playing, and crossing the water. The town has only one government owned general elementary school which encompasses 25 teaching sections within a single compound.

Sample size determination and sampling techniques

The sample size for this study was calculated by using single proportion formula at 95% confidence interval (CI) level (Z (1-ά/2) = 1.96), an expected prevalence of 50% since there was no study conducted regarding this topic in the area and 5% marginal error. Then the sample size was calculated as n = [Z 1-a/2] 2 P (1-p]/d 2 , Where: n = sample size, P = proportion problem in the study area, Z 1-α/2 = CI of 95%, d = Marginal error to be tolerated. By adding 10% of contingency 319 students were included in our study.

The sample size which is determined earlier was used to calculate the proportion of students to be selected from each section. Using registration list, simple random sampling method was employed to select students from each section using a table of random numbers and when the selected student was absent student before or after the indicated one was sampled for replacement.

Socio demography and risk factor assessment

Well structured questionnaire based on the known risk factors for STHs and Schistosoma was locally developed specifically in English version by the research team. It was later translated into the Amharic, local language of the study area. Comparisons were made on the consistency of the two versions. The questionnaires address students' socio-demographic information, hand washing, shoe wearing and swimming habit, presence of latrine and its usage, water source for domestic use and other risk factors around. A pretest was conducted among five percent of the total sample size that was selected randomly from 25 sections on which cross sectional study was conducted by trained data collectors and any ambiguous questions and repetitive ideas were corrected. Additional response categories were also added based on the pretest findings. When the children were identified, the purpose, objectives and benefits of the study were explained to the parents and consent for participation sought. Those who consented were interviewed at the school. All households were inspected to assess the type and condition of latrine, status of the compound, absence or presence of refuse dumps, soiling of the compound and whether the home reared cattle. The type of water sources from which respondents collected water and their accessibility was also noted.

Stool sample collection, processing and light microscope detection

After interviewed the students were then given labeled stool containers with tight covers bearing serial numbers of the subjects and were asked to put about 5 g of stool the following morning. All the stool samples were received the following day at an organized central place and processed using direct saline wet mount for motile intestinal parasites. Stool samples also processed using Kato-Katz smear technique [34]. The slides were examined under light microscope at X100 and X400 magnifications. The parts of the slides was re-examined for assurance of quality control. A small portion of the stool samples were also preserved in 10% formalin for repeating the tests whenever required.

Quality control

For quality purpose Proper thin wet mount and Kato-Katz techniques was prepared using standard amount of specimen and examined within a given time. Ten percent of the slides was randomly selected and re-examined at the end by experienced laboratory technologist who was blind for the first examination result. The result of laboratory examination was recorded on well prepared format carefully and finally it was attached with the questionnaire.

Data management and analysis

After data collection process, the data were checked for completeness. Then the result of laboratory examination was recorded on well prepared format carefully and finally was attached with the questionnaire. The study population was further divided into three age groups, i.e. 5-9, 10-14 and 15-19 years. The egg per gram stool (EPG) was used to categorize intensity of infection. The intensity of infection was classified as low (when EPG was < 200), moderate (EPG = 201-800), and heavy (EPG > 800). The frequency distribution of both dependent and independent variables were worked. The dependent variables were any STH and Schistosoma infections in the children. Potential risk factors explored include sociodemographic characteristics, hand washing, shoe wearing and swimming habits, presence of latrine and its usage and others. The data was entered into a computer using SPSS (15 version) statistical packages and univariate association between each exposure and the presence of infection was assessed using the Chi-squared test and odds ratios with 95% CI were computed as measures of association. P-value less than 0.05 were taken as statistical significant association between dependent and independent variables.

Ethical consideration

Ethical clearance was obtained from University of Gondar, College of medicine and health sciences and department of medical laboratory sciences. Additionally, after explaining the importance of the study briefly an informed written consent was obtained from Parents of the study subjects. Anyone not willing to take part in the study had full right to do so and confidentiality of the study participants was also maintained. Those students who were positive for intestinal parasites were treated accordingly by appropriate antiparasitic drugs.

Results

Socio-demographic characteristics of study subjects

A total of 319 school children (157 males and 162 females) were included in the study. The age range of interviewed children was 5 to 17 years with median of 11 (± 1.2sd) years. The majority 89.4% and 85.9% of students were Amahara by ethnicity and Orthodox Christians by religion, respectively. 70.8% of student's family heads were illiterate and agriculture was their major occupation (76.4%).

Prevalence and intensity of soil transmitted helminths and Schistosoma mansoni

Out of 319 study subjects 263 (82.4%) and 251(78.1%) had one or more parasites in Kato Katz and wet mount techniques, respectively. From soil transmitted helminths, A. lumbricoides was the predominant isolate (22%) followed byhookworms (19%) and T. trichiura (2.5%). S. mansoni was also isolated in 37.9% of the study participants. Though statistically significant difference was not observed in parasitic infection among males and females but infections due to A. lumbricoides and Hookworms appeared relatively higher in males than in females. On the other hand more females than males were affected by T. trichiura and S.mansoni (Table 1).

More than half (53.9%)of children had a single infection, while 23.2% of children had double infections and 5.3% had triple infections. From the infected children, 45.5%, 43.7% and 10.8% harbored single, double and triple parasites, respectively. Higher rate (10.1%) of S. mansoni coinfection with A. lumbricoides was observed followed by hookworms (7.2%) and T. trichiura (0.9%). There was no statistically significant association between multiple infection and sex and age. The rate of heavy infection was highest for A. lumbricoides (14.1%) followed by S. mansoni (7.4%) and Hookworms (3.4%). In contrast no heavy infection was detected due to T. trichiura (Table 2). On the other hand 38.5%, 53.6% and 7.9% of the infected children harboured low, moderate and heavy infection, respectively for different STH and S. mansoni.

Univariate risk factor analysis for soil transmitted helminths and S. mansoni infections

The distributions of the demographic and risk factors of the subjects are shown in Table 3. In univariate analyses, no statistical significant association were observed in parasite prevalence between male versus female, first versus second cycle and maternal educational status of the students. Even though there was no overall statistical significant association between age and parasite prevalence; age group between 5-9 years showed statistical significant association compared to other categories (OR = 3.08; 95% CI 1.017, 9.373, p = 0.047). Similarly significant association in parasite prevalence was observed between family water source (overall p < 0.005) with the highest prevalence was observed in those getting their water from protected spring (89.5%), and the lowest in those using pipe line (74.4%). In addition, frequency of hand washing habit with soap was significantly associated with children infection with increased risk seen for infrequent hand washing habit with soap compared to daily use (overall p = 0.000, OR 0.292, 95% CI 0.130-0.652) ( Table 3).

Separate univariate analysis for specific parasite prevalence and risk factors indicated significant positive associations were seen between presence of Hook worm infection and none-shoe wearing habits(OR = 2.1;95% CI 1.55-4.494, p < 0.001) and S. mansoni infection and those children who had swimming habit in the river (OR = 3.2;95% CI 2.53-8.38, p < 0.001) (data not presented here).

Discussion

Soil-transmitted helminths and schistosomiasis infections are among the most prevalent afflictions of humans who live in areas of poverty in the developing world. Because of the geographic overlap of these afflictions and their impact on children and adolescents, World Health Organization (WHO); the World Bank; and other United Nations agencies and bilateral, and civil society are working to integrate STH and Schistosoma control through a program of periodic schoolbased targeted Antihelminthic drug treatments [16,17].

Despite presence of the deworming program for control of STH and S. mansoni currently in study area, the finding this study showed that over all prevalence STH and S. mansoni was high (82.4%) and the diseases were still a major health problem of the study area. S. mansoni was most prevalent (37.9%,) followed A. lumbricoides, the Hookworms, T. trichiura and any other helminths, 22%, 19%, 2.5% and 1.5%, respectively. The finding of this study is quite high when compared to other studies conducted in Gondar regions and other parts of Ethiopia [23][24][25][26].

A cross sectional survey conducted on STH and S. mansoni infection among school children in Chilga district, North West Ethiopia [22] reported that the over all prevalence rate for one or multiple parasitic infections in children was 68.4% and infection due to A. lumbricoides was the most prevalent (42.9%, range: 22.9%-68.6%)  followed by the hookworms (37.7%, range: 28.0% -65.5 %), S. mansoni (19.4%, range: 7.0% -64.3%) and T. trichiura infection (14.8%, range: 12.7%-20.8%). The differences in prevalence among the different communities appear to be associated with environmental sanitation, water supply and socioeconomic status of households, although this needs to be verified in more extensive follow up studies. Other factors related to macro-and microenvironment, time of study, method of examination, etc, do also contribute to the differences in the prevalence and distribution of these intestinal helminths. No significant difference was obtained in infection rates and egg counts among the age and sex of schoolchildren under consideration. This denotes a similar exposure risk to infection by these helminths.

Other study conducted on intestinal helminthic infections in school children in Adarkay District, northwest Ethiopia, [23] reported overall prevalence rates of 55.3% for S. mansoni, 43.0% for A lumbricoides, 20.2% for hookworm and 11.8% for T. trichiura infections. In similar other studies undertaken in school children the same author has recorded infection rates of 41.3% with 35.0%, 16.5% and 22.8% for A. lumbricoides, S. mansoni, T. trichiura and hookworm infections, respectively, in the Dembia Plains [24]; and 35.6%, 17.3%, 8.5% 3.3% for A. lumbricoides, S. mansoni, T. trichiura and hookworm, respectively, in Gondar town and surrounding areas [25][26][27][28].

When compared with the above studies which were conducted before in the study area and around the study area prevalence of these parasites are still high and being increasing in the study area. This might be due to climatic condition of the Zarima town because climate is an important determinant of transmission of these infections with adequate moisture and warm temperature essential for larval development in the soil [29,30]. Equally important determinants are poverty, inadequate water supplies and sanitation [31]. Therefore, increased prevalence of S.mansoni infection in study area in addition to STH was mainly due to frequent contact of school children for washing, swimming and bathing activities with Zarima River which passes the town. In this study STH and S.mansoni were co-endemic in the study area. The rate of heavy infection was highest for A. lumbricoides (14.1%) and 14.7% of the infected children harbored heavy infection for different STH and S. mansoni. There is evidence that individuals with many helminthic infections have been heavier infections with STH and S.mansoni because morbidity from these infections and rate of transmission are directly related to the numbers of worms harbored in the host [32], and the intensity is mainly epidemiological index used to describe soil transmitted helminthic infection. Also the most striking epidemiological features of human helminthic infections are aggregated distributions in human communities, predisposition of individuals to heavy (or light) infection, rapid re-infection following chemotherapy, and age intensity profiles that are typically convex (with the exception of hookworm).

The high prevalence rate of STH and S. mansoni infections encountered among schoolchildren of the study area raises a serious concern. It signifies the fact that children are the highest risk groups in the community and serve as sources of infection and transmission. These parasites are well known to be associated with lowered work capacity and productivity both in children and adults and increased susceptibility to other infections. Helminths also impair the mental and physical development of children [7,8]. Again the majority of wormy children are not only infected with one species of worm but they also tend to harbor the heaviest burdens and more than two-thirds of children were infected with one or more helminths.

Both host-specific and environmental factors have been identified that may affect the risk of acquiring or harboring heavy intensity helminthic infections. Some of factors are behavior; household clustering, occupation, poverty, sanitation and urbanization have been associated with these helminthic infections [1,2]. The result of this study revealed that age group between 5-9 showed statistical significant association compared to other categories and significant association in parasite prevalence was observed between family water sources. In addition, frequency of hand washing habit with soap was significantly associated with children infection with increased risk seen for infrequent hand washing habit with soap. This finding is almost comparable with other study findings [22,23,26]. The high infection rate among 5-9 age groups is may be due to at this age stage the students have less knowledge about sanitation and more active for swimming and playing on soil. So these factors in addition to other unknown factors might be contributed the high prevalence of STH and S. mansoni among 5-9 years age groups.

The strengthen of this study is that it used standard Kato Katz techniques to detect and count parasites ova, qualified laboratory technologists examined the slide in order to not miss the parasite and the questionnaire assessed many risk factors but the study only used single stool sample which may not address day-to-day and intra-stool variations of egg output that can be the limitation of the study.

Concussions

Prevalence of STH and S.mansoni was quite high and the diseases were still major health problem in the study area. Because of rapid reinfection following chemotherapy, the highly effective drugs now available and the control methods used in study area have not reduced the prevalence of both schistosomiasis and soil transmitted helminths. Therefore, in future other control activities should be focused in addition to deworming program because without improved water supplies and sanitation chemotherapy approach cannot rely on for sustainable reduction in helminthic infection frequency and intensity of infection. There is also a need for community mobilization towards provision of safe and adequate water supply, latrine construction to reduce open field defecation, and health education aimed at bringing behavioral change in the district. Periodic deworming, particularly of the school-aged children with long term improvements of sanitation should be exercised.

Table 1
1Prevalence of STH and S. manson i in Children Attending Zarima elementary School, 2009Parasite s indentified
Male (N = 157)
Female (N = 162)
Total (N = 319)
X 2
P.value

No
%
N o
%
N o
%

S. mansoni
53
33.7
68
42.2
121
37.9
2.28
0.06

A.Lumbricoides
49
31.2
21
12.9
70
21.9
15.5
0.000008

Hookworms
30
19.1
30
18.5
60
18.8
0.018
0.8928

T.trichura
2
1.2
6
3.7
8
2.5
1.925
0.1654

Others
3
1.9
1
0.6
4
1.2
1.077
0.0301

Total
137
87.2
126
77.7
263
82.4

Table 2
2Categorization of Intensity of Infection Due to S. mansoni, A. Lumbricoides, T. trichiura and the Hookworms in School Children in Zarima Town, 2009Infection status
S.mansoni
A.Lumbricoides
Hookworms
T. trichiura

No
%
N o
%
N o
%
No
%

Negative
198
62.1
193
60.5
203
63.6
311
97.5

Low
32
10
20
6.3
39
12.2
6
1.9

Moderate
80
25
40
12.5
19
6
2
0.6

Heavy
9
2.9
10
3.1
2
0.6
0
0

Total positive
121 8
37.9
70
21.9
60
18.8
8
2.5

Total
319
100
319
100
319
100
319
100

Table 3
3Univariate risk factor analysis for soil transmitted helminths and S. mansoni infections among school children in Zarima town, northwest Ethiopia, 2009 STH and S.mansoni in the children (N = 319) Note: STH = soil transmitted helminths, N = total number of study participants, n = number of outcome for each exposure.Variables
N(%)
n(%)
OR (95%CI)
p-value

Demographics variables

sex
Male
157(49.2)
137(87.3)
0.913(0.827,1007)
0.067

Female
162(50.8)
126(77.7)

Age categories

5-9
47(14.7)
43(91.5)
3.08(1.017,9.373)
0.047

10-14
151(47.3)
129 (85.4)
1.68(0.904,3.134)
0.101

15-19
121(38)
91(75.2)
1
0.071

Education level

First cycle(1-4)
160(50.2)
139(86.9)
0.650(0.329,1.093)
0.093
Second cycle(5-8)
159(49.8)
124(77.9)

Mother's education

Illiterate
226(70.8)
191(84.5)
0.764(0.407,1.431)
0.399

Literate
93(29.2)
72(77.4)

Risk factors

Water source

Pipe line
117(36.7)
87(74.4)
1
0.005

Protected spring
153(48)
134(87.6)
0.483(0.196,1.190)
0.114
Well
49(15.3)
42(85.7)
1.427(0.556,3.701)
0.465

Hand washing with soap

Daily
30(9.4)
10(33.3)
1
< 0.001

At least once a week
135(42.3)
112 (83)
0.311(0.011,0.086)
< 0.001

Less frequent than once a week
154(48.3)
141 (91.5)
0.292(0.130,0.652)
0.003

Latrine type

Private toilet
132(41.4)
112(84.8)
1
0.829

Public toilet
30(9.4)
25 (83.3)
0.878(0.325,2.616)
0.878
Open field
157(49.2)
126 (80.2)
1.12(0.384,3.27)
0.836

AcknowledgementsThe authors appreciate the study participants for their cooperation in providing the necessary information and stool sample. We would also like to extend our heartiest appreciation to Zarima town education office and teachers of Zarima elementary school for the cooperation during data collection.Authors' contributions AA conceived the study, undertook statistical analysis and drafted the manuscript. AA, ZA, YS, TT, BM, WB, SG AND BG initiated the study and made major contributions to the study design and statistical analysis. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.Competing interestsAll authors declare that they have no competing financial or any other interest in relation to their work.Pre-publication historyThe pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/11/189/prepub
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Antifungal activity of schinol and a new biphenyl compound isolated from Schinus terebinthifolius against the pathogenic fungus Paracoccidioides brasiliensis

Susana Johann
Nívea P Sá
Luciana Ars Lima
Patricia S Cisalpino
Betania B Cota
Tânia Ma Alves
Ezequias P Siqueira
Carlos L Zani
Antifungal activity of schinol and a new biphenyl compound isolated from Schinus terebinthifolius against the pathogenic fungus Paracoccidioides brasiliensis
R E S E A R C H Open Access
Background: The aim of this study was to isolate and identify the antifungal compounds from the extracts of Schinus terebinthifolius (Anacardiaceae) against clinical isolates of the pathogenic fungus Paracoccidioides brasiliensis. Methods: The hexane and dichlomethane fractions from leaves and stems of S. terebinthifolius were fractionated using several chromatography techniques to afford four compounds. Results: The compounds isolated from S. terebinthifolius were identified as schinol(1), a new biphenyl compound, namely, 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate (2), quercetin (3), and kaempferol (4). Compounds 1 and 2 were active against different strains of P. brasiliensis, showing a minimal inhibitory concentration value against the isolate Pb B339 of 15.6 μg/ml. The isolate Pb 1578 was more sensitive to compound 1 with a MIC value of 7.5 μg/ml. Schinol presented synergistic effect only when combined with itraconazole. The compounds isolated from S. terebinthifolius were not able to inhibit cell wall synthesis or assembly using the sorbitol assay. Conclusion: This work reveals for the first time the occurrence of compound 2 and discloses activity of compounds 1 and 2 against several clinical isolates of P. brasiliensis. These results justify further studies to clarify the mechanisms of action of these compounds.

Background

Paracoccidioides brasiliensis, a dimorphic fungus occurring in Central and South America, is responsible for paracoccidioidomycosis (PCM), an endemic disease that could affect at least 10 million people in Latin America [1]. However, notification of its occurrence is not compulsory in the countries where it is endemic, hence accurate figures regarding the disease incidence and prevalence are difficult to determine [2]. PCM is a pulmonary infection characterized by cutaneous and/or mucosal lesions that can disseminate to other organs [3]. This disease constitutes an important health problem in Brazil and had also been found in Venezuela, Colombia, Ecuador and Argentina [1].

Two forms of the disease have been described in the literature: the acute (subacute) juvenile form and the chronic adult form; the former runs a faster course and is more severe than the latter [4]. However, in both cases, cell-mediated immune functions are abnormal and, in the absence of specific therapy, mortality is high. Even if improvements are observed in patients after specific treatment, lesions usually remain as sequels [5]. The disease may develop either directly from a primary focus with no latency period or, more commonly, by reactivation of quiescent infections [1].

The drugs most commonly used for treating patients with PCM are sulfonamides, ketoconazole, itraconazole, and amphotericin B. Sulfonamides were the first class of drugs available for treating patients with PCM, but long periods of treatment may be required (more than 2 years), with increasing concern about drug toxicity, cost of treatment, and unacceptable rates of noncompliance with therapy [1,6,7]. Unfortunately, amphotericin B has been associated with substantial toxicity. Although itraconazole and ketoconazole are effective against P. brasiliensis, a long duration of therapy and sometimes a lifelong secondary prophylaxis are required, especially in AIDS patients with high relapse incidence [3].

With the aim of discovering new drug leads that could result in new chemotherapeutic or chemoprophylatic agents to combat P. brasiliensis infectitons, our group screened Brazilian medicinal plant's extracts against isolates of P. brasiliensis [8]. Among these plants, Schinus terebinthifolius showed strong activity against clinical isolates of P. brasiliensis. S. terebinthifolius Raddi (Anacardiaceae) is a perennial tree occurring in the Brazilian coast. It is known by a variety of common names, e.g., "aroeiravermelha" and "aroeira pimenteira" [9]. Many biological properties for this medicinal plant were described in the literature: antioxidant and wound-healing [10], antitumoral [9], and antimicrobial activities [11][12][13]8]. We report herein for first time the investigation of natural products isolated from this plant using in vitro assays with P. brasiliensis.

Methods

Maintenance of P. brasiliensis strains

Five clinical P. brasiliensis strains, Pb01 (ATCC-MYA-826), PbB339 (ATCC 32069), Pb18 (Faculty of Medicine, Universidade de São Paulo, São Paulo, Brazil), Pb3 (clinical isolate from chronic PCM, São Paulo, Brazil-MHH Forjaz/TIE Svidzinski), and Pb1578 (clinical isolate PCM, Goiás, Brazil, kindly provided by Dr. Maristela Pereira, Universidade Federal de Goiás) were used in the biological assays. The strains of P. brasiliensis were maintained by continuous passages in YPD (yeast, peptone and dextrose) medium at 37°C. The fungi were used after 7-10 days of growth.

Plant material

The aerial parts of S. terebinthifolius (germplasm bank number 44) were collected at EPAGRI (Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina), Itajaí, Santa Catarina, Brazil, in January 2004.

Extract preparation

The dried leaves (400 g) were extracted by maceration in four liter of ethanol 80% during ten days. After solvent elimination, 36 g of a hydroalcoholic extract (HA-L) was obtained. This extract was then partitioned between water and, successively, with hexane, dichloromethane, and ethyl acetate, resulting in 1.4 g of an hexane fraction (HEX-L), 5.0 g of a dichloromethane fraction (DCM-L), and 6.6 g of the ethyl acetate fraction (EA-L), and 15.2 g of the aqueous fraction (AQ-L). The plant stems (250 g) were submitted to the same procedure resulting in 1.0 g (HEX-S), 3.2 g (DCM-S), 0.8 g (EA-S), and 4.8 g (AQ-S) fractions, respectively [13,8].

Instruments (General procedures)

Silica gel Merck 60G (70-230 mesh) was used for column chromatography and silica gel pre-coated plates Merck 60F 254 were used for thin-layer chromatography (TLC). The spots on TLC were visualized under UV at 254 and 366 nm and also after spraying with vanillin-H 2 SO 4 and heating at 120°C for 10 min. Plates were sprayed with NP-PEG (polyethylene glycol reagent for visualization of flavonoids. Gel filtration was run on columns filled with Sephadex LH-20. Analytical HPLC (high-performance liquid chromatography) were run on a Shimadzu chromatograph equipped with a LC10AD pump, a dual wavelength detector set to 210 nm and 240 nm, and using a Shim-pack C 18 column (5 μm particle size, 4.6 × 250 mm) at a 1 ml/min flow rate. Semipreparative HPLC was run on the same equipment described above using a Shim-pack C 18 (5 μm particle size, 20 × 250 mm) column and 10 ml/min flow rate. Pro-analysis and HPLC grade solvents were purchased from Vetec (São Paulo, Brazil) and Sigma Chemical Co (St. Louis, USA), respectively. The ESI-MS n mass spectra were obtained on an ion trap LCQ Advantage (Thermo Electron, San Jose, CA, USA) mass spectrometer operating in the positive and negative ion modes with an electrospray ionization (ESI) source. The samples were injected directly into the ESI source using the equipment micro syringe. The heated capillary temperature was 200°C, the sheath gas flow rate set to 20 arbitrary units, and spray voltage regulated to 4.5 kV. 1D and 2D NMR (nuclear magnetic resonance) spectra were recorded in CD 3 OD and DMSO-d 6 on a Bruker Avance DRX 400 MHz spectrometer, using tetrametilsylane as internal standard.

Chromatographic fractionation of the extracts HEX-L (1 g) was chromatographed on a column (46 × 3 cm) filled with 60 g of silica gel eluted with mixtures of hexane-methanol of increasing polarity. Seventy-four fractions were collected and tested against P. brasiliensis (isolate Pb18). The fractions 10-15 were grouped (26 mg) and fractionated by RP-HPLC on a Shim-pack C 18 5 μm, 20 × 250 mm column using a gradient of MeOH-water (90-100% of MeOH for 20 min) at flow rate 10 ml min -1 and the absorbance of the effluent measured at 210 and 240 nm. This procedure afforded 15.0 mg of compound 1.

DCM-S (3 g) was injected in a 46 × 3 cm column containing 200 g of silica gel and eluted with a step-gradient of hexane-methanol of increasing polarity. The collected fractions (127) were combined according to their TLC profile in 37 groups. All groups were tested against P. brasiliensis (Pb18) and the majority showed antifungal activity. Group 23 (249 mg) was purified by RP-HPLC in acetonitrile-water gradient from 10 to 50% in 40 min and from 50 to 100% in 10 min, the effluent absorbance was monitored at 220 nm and 240 nm. This experiment yielded three fractions which were subjected to gel filtration in a 30 × 2 cm column filled with Sephadex LH-20 and eluted with MeOH at a flow of 0.5 ml/min. In this way it was possible to isolate 17.0 mg of compound 2 and 15.0 mg of compound 3 and compound 4 (2.6 mg).

Antifungal activity Determination of minimal inhibitory concentrations (MIC)

The bioassay with all isolates of P. brasiliensis was performed following CLSI M27-A 2 guidelines [14] and modifications suggested by Nakai et al. [15] and Johann et al. [8]. Amphotericin B (Sigma, St Louis, USA) and trimethoprim/sulfamethoxazole (SMT/TMP) (Ducto, Brazil) were included as positive antifungal controls. The concentrations of 25-0.03 μg/ml for amphotericin B and 600-1.17 μg/ml for SMT/TMP were used. Their stock solutions were prepared in DMSO (dimethylsulfoxide) and water, respectively, from which twofold serial dilutions were prepared as described in the CLSI document M 27-A 2 [14].

Minimal fungicidal concentrations (MFC)

The in vitro minimal fungicidal concentration (MFC) of each compound tested was determined by streaking 10 μl from each well that showed complete inhibition (100% inhibition or a clear well), from the last positive well (growth similar to that of the growth control well), and from the growth control well onto YPD plates. The plates were incubated at 37°C for 7 days only. The lowest drug concentration at which no colonies were able to grow was taken as the MFC value [16].

Sorbitol protection assay

MIC values were determined for the P. brasiliensis isolate Pb18 using the standard broth microdilution procedure described above. Duplicate plates were prepared: one containing twofold dilutions (from 1000 to 7.8 μg/ ml) of compounds 1 and 2 and the other containing compounds 1 and 2 plus 0.8 M sorbitol. MICs were evaluated after 7 and 10 days of incubation [17].

Checkerboard microtiter test

Eight serial twofold dilutions (from 1000 to 7.8 μg/ml) of compounds 1 and 2 and amphotericin B (from 1 to 0.007 μg/ml) were prepared with the same solvents used in the MIC test. The checkerboard was prepared in microtiter plate for multiple combinations of two antimicrobial agents. Each row (x axis) in the plate contained the same diluted concentration of the first antimicrobial compound; while the concentration in each subsequent row was half this value. Similarly each column (y axis) in the plate contained the same diluted concentration of the second antimicrobial compound; while the concentration in each subsequent column was half this value. The drug combination in which the growth is completely inhibited was taken as effective MIC for the combination [18].

A 100-μl suspension of Pb18, the same isolate used in the MIC test, was added to each well and cultured for 14 days. Fractional inhibitory concentrations (FICs) were calculated as the MIC of the combination of amphotericin B and compounds 1 and 2 divided by the MIC of compounds 1 and 2 or AMB amphotericin B alone. The FIC index was calculated by adding both FICs and this was interpreted in the following manner: a synergistic effect when its value is ≤0.5; no interaction when the value falls within the >0.5 to 4.0 range; and antagonistic effect when it is >4.0. This experiment was also performed to determine the effect of a combination of active compounds with SMT/TMP and itraconazole (Janssen-Cilag) [19][20][21].

Results

The hexane (HEX-L) and dichloromethane (DCM-S) fractions of crude extracts from stems and leaves of S. terebinthifolius were selected for chemical fractionation because they exhibited the strongest activities against isolates Pb01, Pb18, and PbB339 of P. brasiliensis and had not presented any toxicity to murine macrophages [8]. The activity of crude extracts of steam and leaves was noted at 30 μg/ml for the three isolates de P. brasiliensis aforesaid. For fractions HEX-L the activity was noted at 15.2-125 μg/ml for the isolates tested and for DCM-S the activity was at 30 μg/ml. However, the fractions EA-S, EA-L, aqueous steam and aqueous leaves did not present good activity with MIC values of 125-1000 μg/ml for EA fractions and 625-1000 μg/ml for aqueous fractions against isolates Pb01, Pb18, and PbB339 of P. brasiliensis.

After several chromatographic procedures, compound 1 was isolated from the HEX-L fraction of S. terebinthifolius. Compounds 2, 3, and 4 were isolated from the DCM-S fraction. Their spectral data (ESI-MS, NMR, UV) were obtained; analysis and comparison with data from the literature allowed us to identify 1 as schinol; 2 as 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate; 3 as quercetin; and 4 as kaempferol (Additional file 1).

Compounds 1 and 2 ( Figure 1) showed antifungal activity at concentrations ≤250 μg/ml against five isolates of P. brasiliensis (Table 1). Compound 1 displayed activity for the majority of the isolates of P. brasiliensis, with MIC values varying from 7.5 to 125 μg/ml. The flavonols 3 and 4 were inactive at the highest concentration tested (250 μg/ml). This is the first time that the antifungal activity of compound 1 and 2 is described. The Minimal fungicidal activity was identical to CIM for compounds 1 and 2, showing that these compounds have fungicidal activity against P. brasiliensis.

Compounds 1 and 2 were tested on the isolate Pb18 of P. brasiliensis in the presence of sorbitol and showed MIC values similar to those of the medium without sorbitol. Table 2 shows the results of combining known antifungal drugs with compounds 1 and 2. When itraconazole was combined with compound 1, the concentration of the former was halved and that of the latter decreased four times, indicating a synergistic effect (fractional inhibiting concentration index = 0.5) between these substances. No synergy could be observed for the other combinations.

Discussion

In a previous work, our group detected the activity of crude extracts from leaves and stems of S. terebinthifolius against clinical isolates of P. brasiliensis, Cryptococcus neoformans, Sporothrix schenkii, and five clinical relevant species of Candida [13,8]. The antimicrobial activity of the crude extract from the leaves of S. terebinthifolius against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and C. albicans has been reported elsewhere [12].

According to Johann et al. [13] the leaf extract of S. terebinthifolius showed the presence of saponins, flavonoids, triterpenes, steroids, and tannins. Loyd et al. [22]   reported the presence of sesqui-and triterpenes. The tetracyclic triterpene schinol (1) was first isolated by Kaistha and Kier [23]. It was further isolated from other plant species such as Juliana adstringens (Julianiaceae) [24]. We describe here for the first time the isolation and identification of compound 2 (4'-ethyl-4-methyl-2,6,2',6'tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate, for NMR data (Additional file 1). This new biphenyl compound is a positional isomer of 4'-ethyl-4-methyl-2,6,3',5'-tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate, a compound already isolated from fruits of S. terebinthifolius [25]. This was the first report of the isolation of the flavonols quercetin (3) and kaempferol (4) from S. terebinthifolius. However, both compounds have already been described among species of the genus Schinus [26]. When the activity of the compounds isolated in this work is compared to the activity of the extracts and fractions of the S. terebinthifolius [8], it is possible to notice that the activity of the 1 (7.5-62.5 μg/ml) was better than the original fraction HE-L (15.2-125 μg/ml). These results suggest that 1 could be the main active compound, since the other fractions did not present important activity.

The activity of the original fraction of the compound 2 was 15.6-125 μg/ml and the crude extract (DCM-S) was 30 μg/ml, thus, other active compounds could be present and by means of synergism, have greater potency than isolated compounds.

The isolates Pb1578 and PbB339 were more sensitive to compounds 1 and 2, respectively; they were also more susceptible to SMT/TMP when compared with other isolates of P. brasiliensis. Pb01 was the isolate most resistant to compound 1 (150 μg/ml), while isolates Pb18 and Pb 3 were more resistant to compound 2. In the present work we tested fungal isolates of P. brasiliensis of 2 distinct cryptic phylogenetic species: S1 (Pb18) and PS2 (Pb03) [27]. Pb01 and Pb1578 are known as isolates representative of a new phylogenetic species Pb-01-like [28]. Teixeira et al. [28] recommended the formal description of the "Pb-01-like" cluster as the new species Paracoccidioides lutzii.

In the present work, compound 4 (kaempferol) did not present any antifungal activity. However, glucosylated derivates of kaempferol such as 3-O-β-D-glycopyranosyl (1 2)-O-β-D-glucopyranosil (1 2)-O-[α-L-rhamnopyranosyl-(1 6)]-β-D glucopyranoside showed activity against different isolates of Fusarium oxysporum [29]. Yordanov et al. [30] showed that kaempferol is a potential inhibitor of the virulent factors (lipolytic and proteinase activities) responsible for the penetration of C. albicans into human cells.

Báidez et al. [31] have shown that quercetin (3) exhibits activity against Phytophthora megasperma and Cylindrocarpon destructans. However, in the present work, we found that this compound did not present any activity against P. brasiliensis.

Antifungal activity may be due to interference in the cell wall synthesis or assembly. A distinctive feature of compounds acting on the fungal cell wall is that the antifungal effect can be reversed in a medium containing an osmotic stabilizer such as sorbitol. Compounds 1 and 2 were tested on the isolate Pb18 of P. brasiliensis in the presence of sorbitol and showed MIC values similar to those of the medium without sorbitol. This result suggests that these compounds may be exerting their antifungal activity via mechanisms other than the inhibition of the cell wall synthesis or assembly. Thus, new experiments are needed in order to determine the mechanism of action of these compounds against P. brasiliensis.

Use of combined antifungal drugs with different active mechanisms could be a promising therapeutic approach. In our study, we could confirm the antifungal of schinol as well as the synergistic effect between schinol and itraconazole in P. brasiliensis. The synergistic effect between schinol and itraconazole might be clinically useful and valuable because itraconazole has a long duration of therapy [3].

Conclusion

In the present work, the aerial parts of S. terebinthifolius furnished two antifungal compounds active on P. brasiliensis: schinol (1), and new biphenyl identified as 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate (2). These natural products will be further studied to evaluate their toxicity and to elucidate their mechanism of action.

Additional material

Additional file 1: Compounds isolates Dates of TLC, retention time (tR) in HPLC, RMN, UV spectrum and ESI-MS of four compounds isolates.

Figure 1
1Antifungal compounds and chromatograms against Paracoccidiodes brasiliensis solated from Schinus terebinthifolius. Compound 1: schinol; compound 2: biphenyl 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate.

Table 1
1Minimal inhibitory concentrations (MIC) compounds isolated from Schinus terebinthifolius against different isolates of Paraccocidioides brasiliensis (values in μg/ml)Pb18 Pb01 Pb3 PbB339 Pb1578

Compound 1
62.5
125
31.2 15.6
7.5

Compound 2
250
125
250 15.6
250
Compound 3
≥250 ≥250 ≥250 ≥250
≥250

Compound 4
≥250 ≥250 ≥250 ≥250
≥250

Amphotericin B*
0.0062 0.12 0.015 0.0062 0.0062

Trimethoprim-
sulfamethoxazol*

300
300
300 75
75

Compound 1: schinol; compound 2: 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy
[1,1'-biphenyl]-4,4'-dicarboxylate; compound 3: quercetin; and compound 4:
kaempferol. The concentrations of 25-0.03 μg/ml for used for amphotericin B
and 600-1.17 μg/ml for SMT/TMP.

Table 2
2Synergism activity between compounds 1 and 2 with amphotericin B, trimethoprim-sulfamethoxazole and itraconazole against Paracocidioides brasiliensis (isolate Pb18) Johann et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:30 http://www.ann-clinmicrob.com/content/9/1/30Combination
FIC

Compound 2 + amphotericin B
1 (I) 1
Compound 2 + trimethoprim-sulfamethoxazole
1.5 (I) 1
Compound 2 + itraconazole
1 (I) 1
Compound 1 + amphotericin B
1 (I) 1
Compound 1 + trimethoprim-sulfamethoxazole
1.5 (I) 1
Compound 1 + itraconazole
0.5 (S) 1

1

S, synergy; I, no effect; A, antagonism.
Compound 1: schinol, compound 2: 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy
[1,1'-biphenyl]-4,4'-dicarboxylate.

Johann et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:30 http://www.ann-clinmicrob.com/content/9/1/30
AcknowledgementsFunding: This work was funded by Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This work was also supported by FIOCRUZ through a PDTIS grant (RPT-10).Authors' contributions SJ was carried out all experimental work, data acquisition and analysis, literature search and writing the manuscript. NPS was responsible for helpful in assay of antifungal activity. LARSL, BBC, EPS and TMAA involved in characterization of isolate compounds from S. terebinthifolius. PSC and CLZ were responsible for study concept, designing and coordinating the research, supervising the work and revising the manuscript. All authors have read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Enzymes in the rhizosphere of plants growing in the vicinity of the Polish Arctowski Antarctic Station

A Machuca
Department of Plant Science and Technology
Campus Los Ángeles

M Cuba-Díaz
Department of Plant Science and Technology
Campus Los Ángeles

C Córdova
Department of Soils and Natural Resources
Universidad de Concepción
Campus ChillánChile

Enzymes in the rhizosphere of plants growing in the vicinity of the Polish Arctowski Antarctic Station
AA65060E21A84EBE355D7E20DEA7D30AAcid phosphataseβ-glucosidaseureaseColobanthus quitensisDeschampsia antarctica
In maritime Antarctica soil enzymes have been little researched, mainly in the rhizosphere of the only two vascular plants that grow there: Colobanthus quitensis and Deschampsia antarctica.This study evaluated the activities of hydrolytic enzymes β-glucosidase, phosphatase and urease in the rhizospheric soils of C. quitensis and D. antarctica from three different sites in the Polish Arctowski Station in Antarctica.The sensitivity to temperature changes of the enzymes was also evaluated using two incubation temperatures (37 and 12 °C).The highest activities related to the C and P cycles, β-glucosidase and acid phosphatase, respectively, were detected in the rhizospheric soils at the site with the greatest variety of plant cover.Urease activity, related to the N cycle, was high in two of the three sites, and the lowest activity was detected at the site where only D. antarctica was found.The activity of the enzymes decreased when the temperature of incubation was reduced, but the magnitude of the decrease depended on the enzyme.β-glucosidase was the most sensitive to temperature change, whereas urease was least affected.Therefore, temperature changes in rhizospheric soils could lead to changes in substrate degradation rates and consequently in the availability of nutrients (C, N and P) for C. quitensis and D. antartica in maritime Antarctica.

Introduction

Colobanthus quitensis (Kunth) Bartl.(Caryophyllaceae) and Deschampsia antarctica Desv (Poaceae) are the only two vascular plants that have colonized the maritime Antarctic islands (Alberdi et al. 2002).These two species can live in association as a clump of grass with other organisms such as mosses and lichens, or as individuals, and thus the soil rhizosphere could be different.The associations with rhizospheric microorganisms (fungi and bacteria) and the presence of several soil enzymes can play an important role in the adaptation and survival of these plant species in extreme environments like Antarctica.For instance, some hydrolases in the Machuca.et al.

soil like phosphatase, b-glucosidase and urease, are key to the biogeochemical cycles of P, C and N, respectively, contributing to plant nutritional status (Nannipieri et al. 2002;Ali et al. 2015;Gianfreda 2015).Soil enzymes have been used as indicators of microbial activity and the health and quality of soils, mainly in extreme environments, where they show the relative availability or limitation of energy or nutrient sources (Hopkins et al. 2008;Cowan et al. 2014).Most of the extracellular enzymes produced by Antarctic organisms are part of the pool of soil enzymes that efficiently catalyze multiple reactions at low temperatures, and are therefore involved in nutrient cycling.In recent years, the increase in global temperature has resulted in changes in the biological activity of the soils (Steinweg et al. 2013;Burns et al. 2013;Evans and Wallenstein 2014) The responses of enzymes at two different soil incubation temperatures (37 and 12 °C) were also investigated to assess the changes in the catalytic activity of each enzyme for the effect of the temperature change.To the best of our knowledge, this is the first report about enzymes in the rhizosphere of vascular plants in maritime Antarctica.

Materials and Methods.

Soil samples were taken from the plant rhizosphere where the root system of clumps is extended up to around 10 cm depth.Three sites were sampled in the vicinity of the Polish Arctowski Station in Antarctica and each soil sample (AR1, AR2 and AR3) corresponded to a sample composed of rhizospheric soils from four plant clumps (C.quitensis plus D. antarctica or only D. antarctica, depending on the site).The main characteristics of the sites where the soils were collected are shown in Table 1.The sampling was done in the summer (January 2011) when the snow had melted.After collection, the samples were transported at low temperature (5 °C approx.) to the laboratory where they were stored at 4 o C for about two weeks until analysis of enzyme activities.The soil samples were air-dried and sieved through 2 mm mesh to determine potential activities of β-glucosidase (Eivazi and Tabatabai 1988), acid phosphatase (Tabatabai and Bremner 1969), and urease (Kandeler et al. 1999).The analytical determinations were carried out in triplicate and enzyme activities were expressed as mmol g -1 dried soil h -1 .Soil samples were incubated at 37 ºC a standard temperature for enzyme soil assays, and at 12 o C, to explore the effect of temperature on the catalytic rate of the enzymes.

Table 1.Main characteristics of the Antarctic sites where the rhizospheric soils were collected Enzymes in the rhizosphere of plants growing in Antarctica

Results and Discussion

The three AR sites showed different characteristics in the type and abundance of vegetation, with AR1 and AR2 being the most diverse in terms of species types, mainly AR1 (Table 1).At different sites the plant cover might be influencing the structure and size of the rhizospheric microbial communities, which in turn are responsible for the different extracellular enzyme patterns present in soils (Yergeau and Kowalchuk, 2008;Zeglin et al. 2009;Gianfreda 2015).Since the sample size from the rhizosphere was very small, it was not possible to analyze the chemical composition of the soil; however, previous bulk samples taken from AR1 showed high organic matter content (O.M. 9.67%), P-Olsen (53.1 mg.Kg -1 ), and low pH (5.1) (Cuba et al., 2010).

In terms of enzymatic activities in the rhizosphere soils of the AR sites, the highest activities of acid phosphatase and b-glucosidase were detected in soils from AR1 (Figure 1A and B), the site with the greatest variety in the vegetation cover and the farthest from the Arctowski Station and therefore no human traffic.By contrast, the urease activity was high in AR1 and AR2, and very low at the site where only D. antarctica was found (Figure 1C).The β-glucosidase showed the greatest variability between sites and the lowest activity values were detected at AR2 (Figure 1B).

The abundant plant cover and high activity of seabirds at AR1 may be the reason for the high contents of O.M., P and N detected in previous studies (Cuba et al., 2010), which stimulates some enzymes such as urease, xylanase and phosphatases (Beyer et al. 2000;Tscherko et al. 2003).

The urease in the soil is of microbial and plant origin, but it can also come from seabird and penguin excrement (Tscherko et al. 2003).Although the rhizospheric soils at AR2 and AR3 were not chemically analyzed, they would probably have lower nutrient and O.M. contents than AR1 due to the patchy colonization of plant growth and the absence of seabird activity contrasting with intense levels of human disturbance (Table 1).This could be the reason for the low levels of b-glucosidase found at AR2 and AR3 and low level of urease at AR3 (Figure 1B and 1C, respectively).Additionally, D. antarctica was found only in AR3 and this may also be related to the low levels of urease in this rhizospheric soil.On the other hand, the presence of some penguin excrement would explain the level of urease at AR2 (Figure 1C).b-glucosidase belongs to the enzyme complex produced mainly by fungi and bacteria, and it hydrolyses cellulose, contributing to the input of organic C in soils; it is positively correlated with high contents of O.M., low pH and the presence of vascular plants (Hopkins et al. 2008;Zeglin et al. 2009).

Apparently the levels of acid phosphatase and urease in the rhizospheric soils were not as affected by the different characteristics of the AR sites than b-glucosidase.On the other hand, the values of acid phosphatase, b-glucosidase and urease activity determined under standard conditions (37°C) are within the ranges reported by Trasar-Cepeda et al. (2008a,b) for a series of temperate climate soils under agricultural or forest management.

The influence of temperature on enzyme activity revealed that the decrease in incubation temperature from 37º to 12 ºC reduced the enzyme reaction rate as expected.However, the magnitude of this effect depended on the type of enzyme.The enzyme most sensitive to the temperature change was b-glucosidase (68-78% reduction with temperature decrease) and the least sensitive was urease (55-56% reduction).It has been observed in Alpine and Arctic tundra soils that C mineralization is more sensitive to low temperatures than N mineralization, which is a result of different sensitivities of the enzymes participating in the cycles of these elements (Koch et al. 2007;Wallenstein et al. 2009;2011;Jing et al. 2014).Our results suggest that at low temperatures (12 °C) or even lower (like those found in Antarctica), the enzymes must be acting at very low rates, resulting in a slow hydrolysis of the components of the O.M. in rhizospheric soils.

Conclusions

The highest activities of β-glucosidase and acid phosphatase were detected in the rhizospheric soils at the site with the greatest variety of plant cover (AR1).

Urease activity was high in two of the three sites, and the lowest activity was detected at the site where only D. antarctica was found (AR3).The activity of the enzymes decreased when the temperature of incubation was reduced, but the magnitude of the decrease depended on the enzyme.β-glucosidase related to C cycle, was the most sensitive to temperature change, whereas urease related to N cycle, was least affected.

, and in the fragile ecosystems such as Antarctica, changes in temperature can produce changes in the enzymatic activities of soil, which in turn could affect the development of vascular plants and other types of vegetation.The aim of this work was to assess the acid phosphatase, b-glucosidase and urease activities present in the rhizospheric soil of C. quitensis and D. antarctica growing at three different sites of maritime Antarctica.

Therefore, in a global warming scenario an increase in enzyme activities related to the cycles of N, P and C is to be expected in maritime Antarctic soils, which may lead to changes in the availability of nutrients in the rhizospheric soils of C. quitensis and D. antarctica.This is a first approach to explore the sensitivity to temperature changes in the reactions catalyzed by acid phosphatase, β-glucosidase and urease from rhizospheric maritime Antarctic soils.

Figure 1 .
1
Figure 1.Potential acid phosphatase (A), b-glucosidase (B) and urease (C) activities (mmol g -1 soil.h - ) in rhizospheric soils from different Antarctic (AR) sites containing vascular plants, assayed at 12°C () and 37°C (n).The means (±SE) of enzyme activities are shown.

Journal of Soil Science andPlant Nutrition, 2015, 15 (4) 833-838
AcknowledgmentsWe wish to thank Ms. I. Cid and Mr. D. Chávez for technical support, and the Instituto Antártico Chileno (INACH) and Arctowski Polish Station for their logistical support.This research was granted by INACH (Project T_03-09).
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