Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity

Authors


Angela Basilio, Centro de Investigación Básica, Merck Sharp and Dohme de España S.A., Josefa Valcárcel 38, E-28027 Madrid, Spain (e-mail: basilio_angela@merck.com).

Abstract

Aims: To evaluate the patterns of the production of antimicrobial compounds by diverse collection of actinomycetes isolated from different geographies under alternative conditions of pH and salinity in the media.

Methods and Results: Actinomycetes were grouped based on their method of isolation and their phenotype diversity was determined by total fatty acid analysis. A total of 335 representative isolates, including 235 Streptomyces species and 100 actinomycetes from other taxa, were screened for the production of antimicrobial activities against a panel of bacteria, filamentous fungi and yeasts, including some of clinical relevance. Production of antimicrobial activities was detected in 230 strains. In the case of the genus Streptomyces, 181 antimicrobial activities (77% of the tested isolates) were recorded. The activities observed among the other actinomycetes taxa were lower (49% of the tested isolates).

Conclusions: The results of this study support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity present a significant capacity to produce compounds with antibacterial or antifungal activity. The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions.

Significance and Impact of the Study: The results demonstrate that these actinomycetes strains isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity.

Introduction

Actinomycetes comprise an extensive and diverse group of Gram-positive, aerobic, mycelial bacteria that play an important ecological role in soil cycles. Many are well known for their economic importance as producers of biologically active substances, such as antibiotics, vitamins and enzymes (McCarthy and Williams 1992; Sanglier et al. 1996; Horan 1999; Lazzarini et al. 2000). In addition, they are one of the major communities of the microbial population present in soil, and their occurrence is greatly influenced by the environmental conditions of humidity, temperature, pH and vegetation.

Soil actinomycetes for the most part show their optimum growth in neutral and slightly alkaline conditions, and isolation procedures have been traditionally based on this neutrophilic character. Previous works showed the existence of a large diversity of acidophilic actinomycetes that differed morphologically and physiologically from neutrophilic species (Khan and Williams 1975; Williams et al. 1977; Williams and Flowers 1978). Acidophilic isolates grow in the pH range 3·5–6·5, with optimum growth between pH 4·5 and 5·5; while neutrophilic strains grow in the pH range 5·0–9·0 (Khan and Williams 1975). Alkalophilic actinomycetes are also known to occur in soils (Mikami et al. 1982). Many of these isolates, initially assigned to the genus Streptomyces, were later reclassified in the genus Nocardiopsis (Mikami et al. 1985; Korn-Wendisch and Kutzner 1992). Halotolerant actinomycetes grow in salt concentrations above 0·2 mol l−1 NaCl, (Kushner 1985). They have been mostly isolated from marine habitats, as a consequence of their adaptation to the saline environment. Halophily among soil isolates has only been observed in certain genera (Embley 1992).

Historically the most commonly isolated actinomycete genera have been Streptomyces and Micromonospora. As a result, the majority of metabolites identified in screening programmes searching for new antibiotics were derived from a relatively limited pool of organisms (Goodfellow and O'Donnell 1989). The genus Streptomyces is in fact known as one of the major sources of bioactive natural products (Bull et al. 1992; Watve et al. 2001). In the last decade, the intensive screening for new secondary metabolites has also focused on minor groups of actinomycetes, including species that are difficult to isolate and culture, and those that grow under extreme conditions (i.e. alkaline and acidic conditions) (Goodfellow and O'Donnell 1989; Edwards 1993; Lazzarini et al. 2000; Phoebe et al. 2001).

In an effort to improve a screening programme in search of new secondary metabolites with antimicrobial activity, alternative selective conditions of pH and salinity for the isolation of minor groups of actinomycetes not usually recovered in neutral and low osmolarity conditions was tested. The effect of this expanded range of isolation conditions on the patterns of detection of antibiotic activity was then evaluated.

Materials and methods

Isolation of actinomycetes

Soil samples were plated following the dilution plating method on starch casein agar (STC) (Okazaki et al. 1983) supplemented with cycloheximide (1 mg ml−1) and pimaricin (2·5 mg ml−1). Media were adjusted to different pH (5, 7 and 11) after sterilization. Halotolerant actinomycetes were isolated by supplementing the same medium with 5% NaCl. A series of 17 soil samples collected in diverse areas of the Philippines, Spain, Switzerland, Costa Rica, Sri Lanka and Mexico were used for the isolation of actinomycetes tolerant to different conditions of pH and salinity.

Identification of actinomycetes

Actinomycetes were identified to the genus level based on microscopic morphology of the vegetative and the aerial mycelium grown on yeast–malt extract agar (YME) or water agar for 21 days at 28°C. Streptomyces strains were assigned to species-clusters after analysis of their fatty acid composition as determined by gas chromatography.

Culture conditions and extraction procedure

Isolates were grown in complex liquid media containing different amounts of nutrient sources: carbon sources (monosaccharides and disaccharides), complex carbon sources, (wheat flour and soluble starch) nitrogen sources and mineral salts. After different periods of incubation, the broths were harvested and extracted with methanol (González del Val et al. 2001).

Analysis of fatty acid by gas chromatography

Cultures were grown as confluent patches on trypticase soya broth agar (BBL, BBL Microbiology Systems Inc, Cockeysville, MD, USA) at 28°C for 4 days. Vegetative growth was scraped from the surface (100–200 mg) and fatty acid methyl esters (FAMEs) were prepared using a modified sample preparation (Miller and Berger 1985). Analysis of FAMEs was carried out by capillary gas chromatography using a Hewlett-Packard Model 5890 gas chromatograph/MIDI system (MIDI Inc., Newark, DE, USA) equipped with a phenylmethyl silicon column (0·2 mm × 25 m). Chromatography conditions were as recommended by the manufacturer. Individual FAME were identified using the Microbial Identification Software (MIS, MIDI Inc). FAMEs profiles were compared using the Bionumerics software package ver. 2·5 (Applied Maths, Kortrijk, Belgium) and calculating the similarity matrix based on the Pearson product moment. The relationship among the strains was established in by the unweighted pair-group method using arithmetic averages (UPGMA) method and plotted as a dendrogram (Vauterin et al. 1996; Roberts et al. 1998).

Evaluation of antimicrobial activity

In vitro antimicrobial susceptibility tests were performed using a panel which included both clinical pathogens and laboratory control strains. All strains were accessioned in the Merck culture collection and included: three Gram-positive bacteria (Bacillus subtilis MB964, Staphylococcus aureus MB5393 and Enterococcus faecium MB5571), two Gram-negative bacteria (Pseudomonas aeruginosa MB979 and Serratia marcescens MB252), one mycobacterium (Mycobacterium smegmatis MB2233), two yeasts (Candida albicans MY1055 and Saccharomyces cerevisiae W303) and one filamentous fungus (Aspergillus fumigatus MF5668). All bacteria used for the tests, except for B. subtilis, were resistant to at least one known antimicrobial agent: Staph. aureus was methicillin-resistant, Ent. faecium was resistant to vancomycin and β-lactam antibiotics, Myco. smegmatis was resistant to penicillin, aminoglycosides and macrolides; the two Gram-negative bacterial strains were resistant to penicillin, cephalosporins and macrolides, and Ps. aeruginosa was resistant to imipenem (Zak 1980; Davis and Stone 1986; Sunderam et al. 1986; Al-Obeid et al. 1990). Candida albicans was resistant to fungistatic azoles, fluconazole and itraconazole (Carledge et al. 1997).

The inoculum and assay plates for bacteria, yeast and filamentous fungus strains were prepared as described by Suay et al. (2000) and Peláez et al. (1998). Preparation of the samples for filamentous fungi followed the procedures of Arenal et al. (2002).

Aliquots of 100 ml of the seeded agar media were poured into Nunc square plates (24 × 24 cm). The incubation temperature for growing the inoculum and the plates was 28°C (yeasts) or 37°C (bacteria and the filamentous fungi).

Methanol extracts of actinomycetes (25 μl) were applied to the surface of the assay plates seeded with the target micro-organisms. Inhibition zones were measured after 24 h of incubation. Different antibiotics (e.g. amphotericin B, tunicamycin, penicillin G, oxytetracycline, hygromycin B and gentamicin) were used as positive or negative controls in the plates. Methanol (50%) was also used as a negative control. All the antimicrobial activities evaluated in this study showed a diameter of the inhibition zones of the sample of ≥15 mm. An extract that produced an inhibition zone ≥15 mm against one or more of assay strain was scored as ‘active’.

Data analyses

Statistical analyses of the distribution of the antimicrobial activities were carried out using the chi-square test. The actual data were contrasted with the expected distribution frequencies under the null hypothesis, that is, the same proportion of active isolates was considered in each of the taxonomic categories.

Results

A total of 609 strains were isolated from the 17 soil samples in all the selective conditions; the number of isolates recovered from each sample varied widely and ranged from 18 to 316. The highest number of strains was obtained in two different isolation conditions, pH 7 and 11, and the lowest at high salt concentrations (Table 1).

Table 1.  Distribution of actinomycetes included in the study according to their isolation conditions and their origin
LocationNo. of samplesNo. of isolates (isolation conditions)Total
pH 5·0 pH 7·0 pH 11·0pH 7·0 (NaCl)
Philippines793808360316
Costa Rica316425712127
Sri Lanka3163731 0 84
Mexico2111812 1 42
Switzerland114 5 3 0 22
Spain1 410 3 1 18
 Total15419218974609

Among these strains, representatives of the genus Streptomyces were the most frequently isolated group. Members of the families Micromonosporaceae, Nocardiaceae and Pseudonocardiaceae were also identified. Similar numbers of Streptomyces species obtained in the three pH isolation conditions were cultivated for the production of antimicrobial activity. However, in the case of Micromonospora and nocardioform actinomycetes, different number of isolates in neutral, alkaline and acidic pH were included in the study (Table 2). High salt concentrations substantially inhibited the growth of actinomycetes. In fact, halotolerant strains were isolated from only eight of the 17 soils tested. The halotolerant actinomycetes tested were assigned to two major taxonomic groups: Streptomyces, 60 isolates and Nocardiaceae, 12 isolates (Table 2).

Table 2.  Taxonomic distribution of actinomycetes used in this study according to their isolation conditions
FamilyNo. of isolates (isolation conditions)Total
pH 5·0 pH 7·0 pH 11·0pH 7·0 (NaCl)
Streptomycetaceae1161118960376
Nocardiaceae18454112116
Micromonosporaceae122544080
Pseudonocardiaceae246012
Others679223
Total15419218974609

Biological activity

A total of 335 isolates, including 235 strains belonging to the genus Streptomyces and 100 strains of taxa other than Streptomyces, referred to as ‘non-Streptomyces’, were selected to be tested for the production of antimicrobial activity (Table 3).

Table 3.  Distribution of antimicrobial activities in Streptomyces genus and other taxa of actinomycetes (‘non-Streptomyces’)
 GeographyTested isolatesActive isolates (%)
AntibacterialAntifungal
  1. χ2 calculated from the population = 4·66; χ2 theoretical for d.f. = 5 and α =0·05.

StreptomycesPhilippines1577348
Costa Rica316565
Mexico254448
Switzerland154740
Spain77186
Total 2356752
‘non-StreptomycesSri Lanka384526
Costa Rica353423
Philippines225927
Mexico33333
Spain1100100
Switzerland11000
Total 1004526

Extracts from 69% of the isolates, representing 230 strains, showed antimicrobial activity. Soils of all geographical regions yielded actinomycetes-producing activity (Table 3).

In the case of Streptomyces species, a total of 181 strains presented antimicrobial activity (77% of the tested isolates). About 67% (157 strains) of the tested strains produced antibacterial activity whereas 52% (120 strains) showed antifungal activity (Table 3). The group of Streptomyces species from the Philippines produced the highest number of antibacterial activities (73%).

In contrast, the yield of the other actinomycete taxa (‘non-Streptomyces’) was lower and the production of antimicrobial activity was only detected in 49 strains (49% of the tested isolates). Among these isolates, the percentage of antibacterial and antifungal activities was 45% (45 strains) and 26% (26 strains) respectively (Table 3).

The differences between the frequency of activities obtained from Streptomyces species and from non-Streptomycete taxa were not statistically significant (chi-square test, see Materials and methods). Frequencies of activities against the target strains are shown in Table 4. Representatives from the different families and genera showed marked differences in their ability to produce antimicrobial activities. Antimicrobial activity was observed in the five families that were included in the study (Streptomycetaceae, Micromonosporaceae, Nocardiaceae, Pseudonocardiaceae and others) and antibacterial activities against B. subtilis and Staph. aureus were the most frequent. Activities against Gram-negative bacteria were less frequent than against Gram-positive bacteria. It is interesting to note that the highest hit rate was obtained among the representatives of the genus Streptomyces (77% of the tested isolates showed activity). This contrasts with the lower antimicrobial activity levels (48–56%) that was observed among strains of the families Micromonosporaceae, Nocardiaceae and Pseudonocardiaceae.

Table 4.  Distribution of antimicrobial activities by taxonomical group is shown in absolute numbers and the percentage in brackets. The families and the genera are sorted according to the number of isolates tested
FamilyGeneraTested isolatesActive strainsTarget micro-organism
PSESERENTMYCSTAPBACASPCANSAC
  1. PSE, Pseudomonas aeruginosa MB979; SER, Serratia marcescens MB252; ENT, Enterococcus faecium MB5571; MYC, Mycobacterium smegmatis MB2233; STAP, Staphylococcus aureus MB5393; BAC, Bacillus subtilis MB964; ASP, Aspergillus fumigatus MF5668; CAN, Candida albicans MY1055 and SAC = Saccharomyces cerevisiae W303.

StreptomycetaceaeTotal (%)235181 (77)14 (6)15 (6)59 (25)51 (22)114 (49)143 (61)76 (32) 95 (40)16 (7)
Streptomyces2351811415595111414376 9516
MicromonosporaceaeTotal (%)40 19 (48) 1 (2·5) 3 (8) 1 (3) 1 (3)  9 (23) 11 (28) 2 (5)  4 (10) 0
Micromonospora32 13 1 2 0 1  8  8 1  3 0
Actinoplanes8  6 0 1 1 0  1  3 1  1 0
NocardiaceaeTotal (%)39 20 (51) 2 (5·1) 6 (15) 1 (3) 4 (10)  4 (10)  8 (21) 3 (8)  8 (21) 0
Nocardia38 19 2 6 1 4  4  7 3  8 0
Rhodococcus1  1 0 0 0 0  0  1 0  0 0
PseudonocardiaceaeTotal (%)9  5 (56) 0 1 (11) 1 (11) 1 (11)  2 (22)  2 (22) 2 (22)  1 (11) 1 (11)
Saccharopolyspora5  3 0 0 0 0  1  2 1  0 0
Other4  2 0 1 1 1  2  2 2  1 1
OthersTotal (%)12  5 (42) 0 1 (8) 1 (8) 1 (8)  4 (33)  3 (25) 2 (17)  3 (25) 0
Mycelium sterile12  5 0 0 0 1  4  3 2  3 0
Total (%)335230 (69)17 (5·1)26 (8)63 (19)58 (17)133 (40)167 (50)85 (25)111 (33)17 (5)

Table 5 shows the percentage of antimicrobial activities from Streptomyces and other actinomycetes taxa isolated under different conditions of pH and salinity. The halotolerant isolates produced the highest percentages of activities against most of the target micro-organisms used in this study, except for Gram-negative bacteria, that were more frequently inhibited by the strains isolated at high pH. The differences found among the four categories of pH and salinity were statistically significant (chi-square test, P < 0·001). The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions.

Table 5.  Antimicrobial activities of Streptomyces and other taxa of actinomycetes isolated in different conditions of pH and salinity. The highest hit rate observed for each target strain is marked in bold
Isolation conditionsTested culturesActive isolates (%)
PSESERENTMYCSTAPBACASPCANSAC
  1. PSE, Pseudomonas aeruginosa MB979; SER, Serratia marcescens MB252; ENT, Enterococcus faecium MB5571; MYC, Mycobacterium smegmatis MB2233; STAP, Staphylococcus aureus MB5393; BAC, Bacillus subtilis MB964; ASP, Aspergillus fumigatus MF5668; CAN, Candida albicans MY1055 and SAC, Saccharomyces cerevisiae W303.

  2. χ2 calculated from the population = 55·99; χ2 theoretical for d.f. = 12 and α = 0·001; significant difference for null hypothesis P < 0·001.

pH 7116391513334120214
pH 111109111614334417285
pH 598261817455731406
pH 7 NaCl4171024275663464612

Diversity of streptomycetes

Active strains (168) were selected for further characterization on the basis of their fatty acid composition. A dendrogram comparing the 168 active strains was built using the UPGMA method (Fig. 1). The 168 strains were assigned to 15 clusters defined at a cut-off point of 90% similarity. The analysis of the cluster composition identified at least 11 minor groups of strains containing only 26 cultures (11% of the tested strains), many of which were single-member clusters. The composition of the remaining four large clusters (clusters 1, 3, 4 and 11) was heterogeneous in terms of the antimicrobial profiles and the isolation conditions of the strains. The distribution of isolates within each cluster with respect to the isolation conditions is shown in Table 6. Three of the five single-member clusters were isolated in acidic conditions (clusters 5, 7 and 17).

Figure 1.

Diversity of the active Streptomyces isolates based on fatty acid methyl esters analysis. The antimicrobial profiles of the four large clusters is indicated: Gram-positive bacteria (Bacillus subtilis MB964, Staphylococcus aureus MB5393, Mycobacterium smegmatis MB2233 and Enterococcus faecium MB5571), Gram-negative bacteria (Pseudomonas aeruginosa MB979 and Serratia marcescens MB252), yeasts (Candida albicans MY1055 and Saccharomyces cerevisiae W303) and filamentous fungus (Aspergillus fumigatus MF5668)

Table 6.  Distribution of actinomycetes isolates within each cluster defined in Fig. 1 according to the isolation conditions
FAMES cluster numberIsolation conditionsTotal
pH 5·0pH 7·0pH 11·0pH 7·0 (NaCl)
  1. FAMES, fattyacid methyl esters.

 110147435
 232027
 381191139
 47105426
 510001
 612104
 710001
 800011
 940004
1020103
1115891042
1200011
1300101
1410102
1510001
Total54473433168

As expected, the cluster analysis of the antimicrobial profiles indicates that no relationship could be established between the antimicrobial profile and the isolation conditions of the active strains. Relationships about the isolation conditions of the producer strains and the source origin is also shown in the dendrogram (Fig. 2). Within each cluster defined for a specific group of antimicrobial patterns, we can find strains isolated in different pH and salt conditions, and strains obtained from different geographic sources. Strains sharing similar antimicrobial patterns differ in their fatty acid composition and correspond to quite diverse isolates. These data indicated that the hit rates of the isolates obtained in alternative isolation conditions are similar to those produced by Streptomyces isolated at neutral pH. Nevertheless the comparative analysis of the diversity of the strains and their antimicrobial activity patterns revealed that little overlap existed among the isolates obtained in the different conditions and soil sources.

Figure 2.

Figure 2.

Relationship between the taxonomy of the active Streptomyces strains and their antimicrobial activity. The antimicrobial activity profiles (based on the diameter of the inhibition zones) were compared in a dendrogram built using the UPGMA method from a similarity matrix generated with the Euclidian distances. The four different shades of grey in the squares indicate the potency of the antimicrobial activity: from darker/more potency to lighter/less potency

Figure 2.

Figure 2.

Relationship between the taxonomy of the active Streptomyces strains and their antimicrobial activity. The antimicrobial activity profiles (based on the diameter of the inhibition zones) were compared in a dendrogram built using the UPGMA method from a similarity matrix generated with the Euclidian distances. The four different shades of grey in the squares indicate the potency of the antimicrobial activity: from darker/more potency to lighter/less potency

Discussion

This paper describes the effect of different isolation conditions on the selection of actinomycetes. Our analysis of screening for antimicrobial activities showed a high proportion of antimicrobial activities were produced by Streptomyces species. This high frequency of antimicrobial activities among Streptomyces species has been previously observed in other soil and aquatic isolates (Holmalahti et al. 1994; Saadoun et al. 1999; Sponga et al. 1999; Ouhdouch et al. 2001). Futhermore, we observed that the activities against Gram-negative bacteria were less frequent than against Gram-positive bacteria. Saadoun et al. (1999) also found a high percentage of activities against Gram-positive cocci and bacilli and a lower number of activities against Mycobacterium vaccae, Escherichia coli, Aspergillus niger and Candida albicans. Such differences in susceptibility reflect our previous experiences in the screening for antimicrobial products (Peláez et al. 1998; Suay et al. 2000; González del Val et al. 2001). In general, the active isolates showed a wide spectrum of activity against bacteria and fungi. Whether the activities being detected in these cases were due to a single inhibitor acting on multiple microbial species, or mixtures of compounds with different specificities is unclear without chemical fractionation of active extracts. Our data may be skewed by the fact that the Gram-negative strains were highly resistant to many antibiotics and were consistent with the known susceptibility differences among similar target organisms (Peláez et al. 1998; Suay et al. 2000).

The fact that the best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions may reflect the importance of the isolation conditions to uncover less-explored micro-organisms that could produce interesting biological activities.

The evaluation of the diversity of the strains of Streptomyces tested based on the isolation conditions showed that the use of alternative isolation conditions of pH and salinity may enrich for certain groups of species not usually recovered by standard procedures.

In order to evaluate whether the taxonomy of the strains could be correlated with their antimicrobial activity, we performed a cluster analysis of their antimicrobial profiles. This analysis established the relationships among the 168 active Streptomyces strains according to the diversity and the size of the inhibition zones produced against the nine target strains. The results obtained from this comparison agree with our previous experiences (data not shown). As we expected, no relationship between the parameters evaluated was found.

In summary, our results support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity. The potential utility of these actinomycetes in screening programmes for bioactive natural products is confirmed. This ability is not restricted to one family or genus within actinomycetes, but rather, all of them offer opportunities to obtain bioactive compounds.

Acknowledgements

The authors are grateful to the CIBE-MSD technicians involved in this study for their technical assistance, notably for their help with the assays, the preparation of the samples and the fermentations. In addition, we thank G.F. Bills for his critical review of the manuscript.

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