Issue 4, April 2004
Biological & Biomedical Sciences
Searching for narrow spectrum antibiotics from microbes in soil from Presque Isle, Pennsylvania
Diana R. Cundell1, Ph.D., and Bryan Brendley2, Ph.D.
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Growth in antibiotic resistance has meant pharmaceutical companies
are now looking to develop novel narrow-spectrum therapeutics whose
specificity is paralleled by decreased side effects. As such antibiotics
might be secreted by bacteria living in ecosystems with few competitors,
we characterized the soil microbiology and chemistry of twenty separate
sites on the sandy peninsula of Presque Isle, Lake Erie, Pennsylvania,
to try and identify biotic or abiotic factors that might promote
the recovery of these species. Twelve individual antibiotic bacterial
isolates were recovered from six coastal and one inland site, and
their recovery was directly correlated with elevated soil fungal
and staphylococcal levels (p < 0.0001). No biotic factor was
associated with recovery of the five isolates secreting narrow-spectrum
antibiotic activity; although, coliform numbers were higher at these
five locations (p = 0.06). Sites from which bacteria with broad-spectrum
antibiotic activity were isolated possessed significantly higher
fungal numbers than those where antibiotic-producing species were
absent (p < 0.001). These data suggest that Presque Isle fungal,
staphylococcal and antibiotic-producing bacteria occupy similar
niches. No single biotic or abiotic factor appears to influence
the spectrum of antibiotic activity produced by bacterial isolates,
which may instead be a response to a complex mixture of variables.
All single specificity antibiotic activities are now being evaluated,
using microbial assays, to be followed by chemical purification
Antibiotics first became widely available in the 1940s with the
use of penicillin and sulfonamides. Since that time, the pharmaceutical
industry has developed more than 100 varieties of these drugs, with
150 million prescriptions being written for antibiotics annually
in the United States alone (Levy 1998). This growth in antibiotic
usage has been paralleled by the ability of bacteria to resist being
killed by these agents, and has resulted in a steady decline in
the number of effective antibiotics each year (Levy 1998). At its
most extreme, the acquisition of antibiotic resistance genes has
resulted in at least four species of bacteria for which there are
no effective forms of conventional therapy available (Levy 1998;
Talaro and Talaro 1996; Jensen and Wright 1997). In order to combat
these infections, new antibiotics will need to be developed to which
bacteria are less likely to become resistant. One approach taken
by many pharmaceutical companies is to focus on the identification
of antimicrobials with narrow specificities restricted to a single
genus or species rather than the broad-spectrum approaches of the
past (Mincey 2001).
Antibiotic production is a feature of several kinds of soil bacteria
and fungi and may represent a survival mechanism whereby organisms
can eliminate competition and colonize a niche (Talaro and Talaro
1996; Jensen and Wright 1997). We hypothesized that hostile environments
might be colonized by narrow-spectrum antibiotic-producing organisms,
since they would face an existence competing for nutrients with
only a few other species. If this were the case, the converse should
also be true: that soils with normal or elevated levels of nutrients
might generate a more competitive environment which would favor
species able to secrete antibiotics effective against a variety
of different bacteria (i.e., broad-spectrum) rather than those with
Isle, Lake Erie, in Northern Pennsylvania, possesses a unique diversity
of native animal and plant species (Discover Presque Isle Park Info)
but has a microbial population that is not well understood. Since
this location is predominantly sand with some loam, coastal areas
where the soil has not been supplemented with either humus or fertilizer
would be expected to be fast-draining and nutrient-poor with only
a few dominant microbial species. Such locations might yield bacteria
secreting narrow-spectrum antibiotics directed against the few microbes
able to survive these hostile environments. Conversely, inland trails
and tourist attractions, which have been landscaped and fertilized,
would be more likely to possess a diverse population of microbes,
including bacteria producing broad- rather than narrow-spectrum
test these hypotheses, we investigated the microbial flora at twenty
sites in and around the Presque Isle area, which included coastal
sites on the Presque Isle bay, inland trails, and points of scenic
interest, at the end of June 2002. Although both fungal and bacterial
species are known to produce antibiotics, fungi tend to produce
mostly broad-spectrum activities, and, in addition, far more antibiotics
are produced by bacteria (Salyers and Whitt 2001). Our study focused
instead on the recovery and isolation of bacterial species able
to produce narrow-spectrum antibiotics, which are primarily in the
hardy, spore-producing Bacillus and Actinomyces
genera (Atlas 1995). To characterize the type of microhabitat favored
by both narrow- and broad-spectrum antibiotic-secreting bacteria,
chemical analyses of the soil for nitrogen, phosphorus, potassium,
humus, and pH were made in addition to enumeration of the total
numbers of bacteria, fungi, yeasts, staphylococci, and coliforms
(both environmental and fecal), using standard microbiological techniques.
Topsoil samples to a depth of four inches (10 cm) were obtained
from 20 separate sites in the Presque Isle area (Table 1). For each
site, the GPS coordinates, presence or absence of earthworms, and
plant flora were noted. Dominant plants at each collection site
were visually assessed and post-emergent leaves were verified using
a comprehensive field manual (Rhodes 2000). Samples were stored
individually in separate plastic containers, refrigerated, and processed
for soil chemistry and microflora (within 72 hours).
1. Soil and Location Characteristics of Presque Isle Samples
(Click to view enlarged table)
Nitrogen, phosphate, potassium, pH, and humus levels for the soil
were assessed semi-quantitatively using commercially-obtained test
kits (Luster™ Rapitest™ Soil Kit).
and fungal levels
The microbial content of the soils was determined by plating 100
µL of 10-2 suspensions in sterile saline, using nutrient,
Sabouraud-dextrose MacConkey, and mannitol agars (Tortora 1998).
Total counts of all non-fastidious bacteria and mycetes were determined
from the nutrient agar plates, with colonial morphology, and microscopic
appearance following staining being used to differentiate between
bacteria, yeasts, and molds. Sabouraud-dextrose agar was used to
determine total fungal numbers. The remaining two agars were differential:
MacConkey agar allowed for differentiation between environmental
coliforms and non-coliforms, and mannitol salt between pathogenic
and non-pathogenic staphylococci. All plates were incubated at 30°
C. The number of colonies appearing after 48 hours was determined
by direct visual assessment. Each soil sample was replicated a minimum
of three times to determine consistent bacterial and fungal numbers.
Fecal coliforms were differentiated from environmental coliforms
according to Geldreich (1965). To differentiate Staphylococcus aureus
from other staphylococcal species, a coagulase slide test using
rabbit plasma was employed according to Pierce (1999).
and analysis of activity of antibiotic-producing isolates
Actinomycete/Bacillus species were isolated by the embedded agar
method described by Atlas (1995). A panel of four commercially obtained
human bacterial pathogenic strains, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, was used
as the test bank. Antibiotic production by a colony was defined
as the inhibition of embedded bacterial growth by a ≥ 3 mm ring
around the soil bacterial isolate on at least six separate occasions.
Narrow-spectrum activity was putatively defined as that inhibiting
one of the four human pathogens, with broad-spectrum inhibiting
two or more. Soil isolates impairing the growth of one or more human
pathogens were putatively identified as either Actinomyces or Bacillus
species by standard microbiological techniques and the Bacilli typed
using a commercial miniaturized API 50CHB test system (Biomerieux-Vitek,
Hazelwood, MO) (Schraft et al. 1996).
Bivariate correlation statistics using the Pearson coefficient were
performed (SPSS for Windows Version 6.13, 2001) to determine the
relationship between microflora levels at the 20 Presque Isle sites.
Comparison statistics of the distribution of narrow- and broad-spectrum
antibiotic producers relative to other microflora, and of coastal
versus inland microflora, were made using nonparametric Wilcoxon
Signed Ranks Test and parametric paired T-tests, respectively (Minitab
for Windows 2000 Version 5.0, 2002).
of narrow- and broad-spectrum antibiotic-producing bacteria
Of the 12 antibiotic-producing bacterial isolates, 11 were Actinomyces
species with the 12th (recovered from site 20) being a Bacillus.
The distribution of antibiotic-producing bacteria per se
was found to strongly correlate with those of fungi and staphylococci
(p < 0.001, r = 0.68 and 0.58, respectively) and total bacterial
counts (p < 0.04, r = 0.47). Comparison of sites from which narrow-
and broad-spectrum antibiotic-producing bacteria were isolated demonstrated
no statistically significant differences in nitrogen (20.0 ±
5.47 and 25.0 ± 5.0 ppm) or phosphate (17.5 ± 2.5
and 27.0 ± 9.7 ppm) levels. Levels of yeasts and total bacterial
counts were also closely similar at the two groups of sites, although
fungal, coliform, and staphylococcal counts showed significant variation
(Figure 1). These differences were significant only in the case
of bacteria secreting broad-spectrum antibiotic activity recovered
from sites with significantly (p < 0.001) higher levels of fungi
than those from which antibiotic-producing bacteria were absent.
No biotic factor was associated with recovery of the five isolates
secreting narrow-spectrum antibiotic activity although coliform
numbers were higher at these five locations (p = 0.06) (Figure 1).
1. Differential soil microflora at sites from which bacteria
secreting narrow- and broad-spectrum antibiotic activity were
recovered, compared to remaining sites. Values shown represent
the mean of three experiments, where 100 µl of a 10>-2
dilution of soil in sterile saline was plated, and results
are given as CFU/ml ± SEM. Data indicated with an asterisk
(*) are significant at p < 0.001.
Specificity of antibiotic-producing soil isolates
Analysis of the antibiotic activities released by the 12 isolates
recovered demonstrated that five were narrow-spectrum, with activities
primarily directed against P. aeruginosa and S. aureus,
and that the remaining seven broad-spectrum activities were primarily
directed against P. aeruginosa, B. megatherium,
and S. aureus (Figure 2).
2. Spectra of antibiotic activity displayed by Actinomyces
and Bacillus soil isolates from Presque Isle, PA.
Data in the graph are representative of the inhibition of
the human pathogenic bacterial strain tested by the soil isolate
of > a 3 mm diameter on at least six separate occasions.
“Narrow-spectrum” (five isolates) indicates that
activity was directed against a single bacterial pathogen,
and “broad-spectrum” represents inhibition of
two or more test bacteria.
chemistry and plant species
All sites examined showed overall similar pH (5.5-6.0) and adequate
to elevated levels of potassium (750.0 ± 34.4 ppm). Levels
of nitrogen and phosphate for the 20 soil sites ranged from 0 to
50 ppm (21.5 ± 3.0 ppm and 22.8 ± 3.3 ppm, respectively
[overall mean ± standard error of mean (SEM)]; Figure 3).
Inland sites were all similar, possessing adequate levels of both
nitrogen and phosphate (33.3 ± 10.3 and 25.0 ± 12.2
ppm, respectively; Figure 3). Coastal locations showed the most
variability, with three separate distributions of nitrogen and phosphate
levels observed (Figure 3). Normal levels of both nitrogen and phosphate
were observed at five locations (sites 1, 3, 4, 19 and 20; 29.0
± 4.0 and 32.0 ± 7.3 ppm for nitrogen and phosphate,
respectively). Low levels of both nitrogen and phosphate were observed
at a further four sites (sites 2, 6, 7 and 18; 10.0 ± 0 and
8.7 ± 1.3 ppm for nitrogen and phosphate, respectively).
Four other coastal sites had low levels of nitrogen but not phosphate
(sites 8, 9, 10 and 17; 7.5 ± 2.5 and 27.5 ± 7.5 ppm
respectively). The remaining coastal site (site 5) possessed adequate
nitrogen (site 5; 40 ppm) and reduced phosphate levels (10 ppm).
nitrogen and phosphate levels of costal and inland sites at
Presque Isle, PA. Data in the figure indicate the number of
coastal and inland sites tested that showed either low (<
10 ppm) or adequate (20-40 ppm) levels of nitrogen (N) or
phosphate (P) as assessed using Luster chemical kits. Combinational
sites indicate those at which one nutrient was adequate and
the other was low or depleted.
of soil microflora
All sites sampled contained bacteria (54.9 ± 7.5 x 103
CFU/ml) and yeasts (20.1± 4.9 x 103 CFU/ml) with
staphylococci present at 18 of the 20 sites (11.8 ±
6.1 x 103 CFU/ml). At four sites (sites 3, 4, 5 and 7)
Staphylococcus aureus was the predominant staphylococcus.
Fungi were isolated from 13 of the 20 sites and were at comparable
levels with the staphylococci in these areas (15.3 ±
4.3 x 103 CFU/ml) (Figure 4). Coliforms were found at
10 sites; three of which were fecal coliforms present at elevated
levels (sites 3, 4 and 5) (Figure 4).
4. Soil microflora of coastal and inland sites at Preque
Isle, PA. Values shown represent the mean of three experiments,
where 100 µL of a 10-2 dilution of soil in sterile saline
was plated, and results are given as CFU/mL ± SEM.
of soil microflora
Recovery of fungal isolates was directly correlated with that of
bacterial species per se (p < 0.02, r = 0.47) and staphylococci
(p < 0.001, r = 0.91). In contrast, no correlation was observed
between the number of yeast colonies or coliforms at the 20 sites
or any other microbe investigated (p ≥ 0.3, r = 0.3). Fungi
were absent from all four sites with low or depleted levels of nitrogen
and phosphate and from seven of eight sites with low to depleted
levels of nitrogen alone (adequate phosphate) (p < 0.001). Adequate
to sufficient levels of nitrogen and phosphate (20-50 ppm) correlated
with the presence of fungal species at 10 of the 11 sites (p <
0.001). No other correlations between nutrient availability and
microbial species were observed.
In this study,
we have shown for the first time that in the temperate, sandy, Presque
Isle area both narrow- and broad-spectrum antibiotic-producing bacteria
occupy niches with similar biotic characteristics. Although our
initial hypotheses were that broad-spectrum antibiotic-secreting
bacterial isolates would occupy microbially diverse habitats with
narrow-spectrum activities favored by more bacterially restricted
habitats, they were not supported by the data from this study. Total
prokaryote numbers at sites with broad- and narrow-spectrum antibiotic-secreting
bacteria were closely similar (Figure 1). Sites with narrow-spectrum
and broad-spectrum antibiotic-producing bacteria also demonstrated
no statistically significant differences in nitrogen (20.0 ±
5.47 and 25.0 ± 5.0 ppm) or phosphate (17.5 ± 2.5
and 27.0 ± 9.7 ppm) levels. In addition, similar numbers
of broad- and narrow-spectrum antibiotic-secreting isolates (seven
and five, respectively) were recovered and shared occupancy of two
of the seven recovery sites.
numbers were the only biotic factor found to statistically distinguish
sites with narrow-spectrum and broad-spectrum antibiotic-producing
bacteria, being elevated at all sites from which broad-spectrum
antibiotic-producing bacteria were recovered compared with sites
from which they were absent (Figure 1). Most fungi generate a variety
of factors with broad-spectrum antimicrobial-activity that limits
bacterial numbers in a particular ecosystem (Salyers and Whitt 2001),
so a similar distribution with broad-spectrum antibiotic-producing
bacterial organisms is not entirely unexpected. Actinomycete, Bacillus,
and fungal species are also able to survive in drier soils provided
by a sandy ecosystem through the generation of spores, which allow
for colonization of a greater range of habitats, especially sandy
and/or dry locations (Mishustin 1975). In addition, fungi possess
a variety of enzymes that are released from cells to externally
break down otherwise inaccessible substrates such as chitin and
lignin (Ljungdahl and Eriksson 1985), therefore providing a microenvironment
that would be comparatively nutrient rich. A study by Griffin (1969)
suggested that in drier, humus-poor soils, fungal mycelia extend
further, pulling water back to the main body of the microbe and
producing a microenvironment that allows more resilient species
of bacteria to grow. In addition, the Presque Isle soil pH of 5.5-6.5
would be expected to favor colonization by cellulose-degrading fungi
whose activities are most active at an acid-neutral pH (Atlas and
antibiotic-producing bacteria were more likely to be isolated from
sites where coliforms but not staphylococci were elevated (p = 0.06,
Figure 1). Three of these locations (sites 3, 4 and 8) were only
colonized by antibiotic-producing bacteria with narrow-spectrum
activities and were also the only sites from which fecal coliforms
were recovered. Shorebird migration is incremental from April onwards,
peaking in August, with sites 3 and 8 constituting major bird-watching
areas in the park (Discover Presque Isle Park Info.) Although coliforms
are rapidly eliminated in marine habitats by predation and competition
(Mitchell 1968), the associated biological wastes they inhabit may
provide transient nutrient sources and alter the pH of the microenvironment
to create a suitable habitat for Actinomyces species. Given the
closeness to statistical significance of the data (p = 0.06), despite
the fact that very few antibiotic-producing isolates per se were
identified, it is anticipated that further sampling and analysis
of coliform-rich coastal soils should allow us to identify the relationship
between these two groups of bacteria.
The low levels
of antibiotic-producing bacteria isolated in the current study may
have several contributing factors. The sandiness of the soil allowed
easy drying and several species could have been lost during transport.
In addition, sampling of sandy-loam temperate soils during late
spring (May) has been previously shown to recover a bacterial biomass
only around half that of late summer (August) (Neher et al. 1999).
Since antibiotic-producing organisms are often the least prevalent
bacteria in these ecosystems (Mishustin 1975), this could also significantly
affect our results. Actinomyces species are more prevalent in soils
with a neutral to alkaline pH (Hattori and Hattori 1976), so the
acidic environment (pH 5.5-6.5) of Presque Isle would restrict colonization
to the more resilient members of this genus. This is also the case
for Bacilli, whose endospores are generally well-distributed across
a variety of soils with active bacteria, usually being recovered
from soils with alkaline or extremely alkaline culture conditions
(7.0-9.0) (Kroll 1990). Another issue is that some endospores present
in the samples may also have been unable to be cultured under laboratory
conditions using standard microbiological methods, similar to more
than an estimated 99% of environmental organisms (Pace 1996).
current study was unable to successfully separate the biotic or
abiotic factors influencing narrow- or broad-spectrum antibiotic-producing
bacteria to colonize a habitat, certain factors were identified
that appeared to favor the presence of both these species. First,
all were isolated from sites that were primarily coastal (6/7 locations)
but with adequate levels of nitrogen (6/7 locations) and/ or phosphate
(5/7). Secondly, strongly positive correlations were observed between
antibiotic-producing bacterial isolates per se and both total fungal
and staphylococcal counts (p < 0.001, r = 0.68 and 0.58, respectively).
Sessitsch et al. (2001) observed that amending sandy soils with
organic components had no effect on the overall bacterial distribution,
which was related instead to the overall size of soil particles
that the microbes inhabited. These data seem to support our conclusions
since coastal and inland sites showed little overall difference
in either microbial numbers (Figure 4) or nutrient levels (Figure
3). Coastal and inland sites at Presque Isle do however differ physically
from one another. Due to high levels of erosion, the lakeside of
the Presque Isle peninsula is annually supplemented with tons of
lake-dredged sand. These sandy soils are low in nutrient content
and have sparse vegetation (Table 1) but are also fresher, more
disturbed, and constantly changing ecosystems. Inland soils, though
sandy, are older and more consistent, characterized by a surface-covering
of moss, established grass, and upland hardwood tree ecosystems
of oak or maple. These observations may therefore suggest that the
antibiotic-producing bacteria are more likely to be recovered from
disturbed habitats than established ecosystems.
to understanding the distribution of the Presque Isle antibiotic-producing
organisms is to examine their spectrum of antibiotic activity. Eight
of the 12 possessed activity against P. aeruginosa, and two produced
narrow-spectrum antibiotic activity (Figure 2). The three remaining
isolates had antimicrobial activities directed against B. megatherium
(2/5 isolates) and S. aureus (1/5 isolates), with none being recovered
that specifically impaired E. coli (Figure 2).
studies have suggested that the distribution of bacterial species
in sandy soils is limited with predominant species from the a-Proteobacteria
group (Sessitsch 2001), which includes certain species of Pseudomonas
(Krieg 1984). Pseudomonas distribution was not documented in this
study, but these species are known to be present in most moist soil
and water environments (Black 1996). The sandiness of the Presque
Isle samples might not allow sufficient water retention inland,
but, at coastal sites, which receive regular moisture, these species
might be expected to compete with resident antibiotic-producing
bacteria for nutrients. In contrast, the direct correlation of antibiotic-producing
bacterial distribution with that of staphylococci was surprising,
since only four of the 12 antibiotic-producing Actinomyces
and Bacilli species impaired the growth of S. aureus in our study
(Figure 2). These data suggest that, unlike Pseudomonas species,
Actinomyces, Bacilli, and staphylococci share the ecosystem.
No single isolate possessed narrow-spectrum activity against E. coli, although three were found that inhibited both the growth of
our test human pathogenic strain, together with P. aeruginosa (Figure
2). Several factors might explain these data. Both E. coli and P. aeruginosa are gram-negative bacilli, which would make them equally
susceptible to a cell-wall-directed antibiotic. Another factor that
supports a “shared” target for E. coli comes from the
finding that it is rapidly cleared from coastal habitats, often
within 24 hours (Mitchell 1968). As it would not play a major role
in these habitats, being a transient occupant, indigenous microbes
would not target this bacterium as a potentially competitive species.
study has therefore demonstrated, for the first time, that both
narrow- and broad-spectrum antibiotic-producing bacterial species
may be recovered from the temperate, sandy Presque Isle ecosystem.
We have also shown that the biotic factors involved in the generation
of a hospitable ecosystem for Actinomyces and Bacillus
species at this site are the presence of fungal and staphylococcal
species together with adequate soil nitrogen levels, but were unable
to effectively identify a set of environmental factors selecting
for narrow-spectrum antibiotic activity. We are planning to re-visit
the Presque Isle site again this summer, to extend the data from
the current study by increasing the number of sites sampled, determining
additional constituents of the microbial flora (including Pseudomonas
species), and measuring soil levels of the microbial micronutrients
iron, zinc, and calcium. To determine whether narrow-spectrum antibiotic
production is favored by a more subtle interplay between several
variables, we also plan to employ artificial intelligence programs
to create a “model” of the Presque Isle environment.
Although the antibiotic moieties described in this study have not
yet been characterized, all single specificity Actinomyces
isolates are now being further evaluated for their spectra of activity,
using species identification and chemical purification of activity.
The authors would like to acknowledge Michael P. Piechoski for
his technical assistance in the execution of these experiments,
and Dr. William H. Brendley for his support and advice in preparation
of this article. Funding for the project came from a Pennsylvania
SEA Grant and summer research grants from Philadelphia University
and Gannon University. Permission for soil collection from the Presque
Isle site was under an agreement with the Pennsylvania Department
of Conservation and Natural Resources (DCNR)
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