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Effect of antibiotics in the environment on microbial populations

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Appl Microbiol Biotechnol (2010) 87:925–941
DOI 10.1007/s00253-010-2649-5
MINI-REVIEW
Effect of antibiotics in the environment
on microbial populations
Chang Ding & Jianzhong He
Received: 2 February 2010 / Revised: 24 April 2010 / Accepted: 25 April 2010 / Published online: 28 May 2010
# Springer-Verlag 2010
Abstract Antibiotics act as an ecological factor in the
environment that could potentially affect microbial com-
munities. The effects include phylogenetic structure alter-
ation, resistance expansion, and ecological function
disturbance in the micro-ecosystem. Numerous studies have
detected changes of microbial community structure upon
addition of antibiotics in soil and water environment.
However, the causal relationship between antibiotic input
and resistance expansion is still under debate, with evidence
either supporting or declining the contribution of antibiotics
on alteration of antibiotic resistance. Effects of antibiotics on
ecological functions have also been discovered, including
nitrogen transformation, methanogenesis, and sulfate reduc-
tion. In the latter part, this review discusses in detail on factors
that influence antibiotic effects on microbial communities in
soil and aquatic environment, including concentration of
antibiotics, exposure time, added substrates, as well as
combined effects of multiple antibiotics. In all, recent research
progress offer an outline of effects of antibiotics in the natural
environment. However, questions raised in this review need
further investigation in order to provide a comprehensive risk
assessment on the consequence of anthropogenic antibiotic
input.
Keywords Antibiotics . Resistance gene .
Microbial community . Soil . Aquatic environment
C. Ding : J. He (*)
Division of Environmental Science and Engineering,
National University of Singapore,
Block E2-02-13, 1 Engineering Drive 3,
Singapore 117576, Singapore
e-mail: jianzhong.he@nus.edu.sg
Introduction
Antibiotics are either naturally occurring or man-made
chemicals that can be divided into different classes such as
ß-lactams, quinolones, tetracyclines, macrolides, and sulfo-
namides (Kümmerer 2009a) (Table 1). The main source of
antibiotics in the environment is anthropogenic input from
wastewater discharge, manure disposal, and aquaculture
(Kümmerer 2009a), as is supported by the correlation of
human activity with the occurrence of antibiotics in the
natural habitat (Yang and Carlson 2003). Once antibiotics
enter the ecosystems, they can be treated as an ecological
factor, driving the evolution of the community structure
(Aminov and Mackie 2007). Accordingly, the change of
community structure influences the ecological function of
soil and water ecosystems such as biomass production and
nutrient transformation (Thiele-Bruhn and Beck 2005;
Kotzerke et al. 2008). Indirect effects from the antibiotic
disturbance to the micro-ecosystem are largely unknown,
and it is expected that such disturbance might have
significant and long-term effects on the rate and stability
of ecosystem functioning (Perry et al. 1989). Therefore, this
review summarizes current knowledge of the effects of
antibiotics on microbial community in natural habitat.
Though the inhibition effects of antibiotics on microbes
have been reported in the reviews by Halling-Sørensen et
al. (1998) and Thiele-Bruhn (2003), those studies primarily
focus on eukaryotic organisms, pure cultures, or on general
effects like growth inhibition and acute toxicity on
microbial community. In order to provide an update on
current knowledge of antibiotic effects, this review empha-
sizes on how antibiotics alter the microbial community
structure, inhibit or promote ecological functions, and affect
the magnification of resistance, mainly in the context of the
natural environment. Antibiotic concentrations, exposure
926 Appl Microbiol Biotechnol (2010) 87:925–941

Table 1 Summary of antibiotics spectrum and mode of action (Halling-Sørensen 2001; Kahn and Line 2005; Beers 2006)

Antibiotic class Example Gram-positive Gram-negative Bactericidal action Bacteriostatic action Mode of actiona

Aminoglycosides Kanamycin X X A
Streptomycin
β-lactams Amoxicillin X (X) X B
Ampicillin
Benzylpenicillin
Penicillin
Glycopeptides Vancomycin X X B
Macrolides Erythromycin X X A
Tylosin
Polypeptides Bacitracin X (X) X B
Colistin X X C
Polymyxin B X X C
Quinolones Ciprofloxacin (X) X X D
Flumequine
Ofloxacin
Oxolinic acid
Sulfonamides Sulfachloropyridazine X X X E
Sulfadiazine
Sulfamethoxazole
Sulfapyridine
Tetracyclines Chlortetracycline X X X A
Doxycycline
Oxytetracycline
Tetracycline
Others Chloramphenicol X X X A
Clindamycin X (X) X A
Lincomycin X (X) X A
Novobiocin X (X) X D
a
Antibiotic mode of action: A inhibition of protein synthesis, B inhibition of cell wall synthesis, C impairment of cell membrane function, D inhibition of
DNA synthesis and replication, E inhibition of folinic acid and consequently DNA synthesis

time, substrate, and multiple antibiotics are also discussed
in detail on their influences on microbial communities in
the natural habitat.
Effects of antibiotics on microbes in soil
Soil plays a vital role in ecosystems, serving as the
primary nutrient base and habitat for plants and organisms,
and functioning as a giant bioreactor for degrading
pollutants and facilitating nutrient transformation. Inevi-
tably, soil is a hot spot for antibiotics to affect indigenous
microbes since it receives a large portion of excreted
antibiotics through application of manure and sewage
sludge as fertilizers (Thiele-Bruhn 2003). Also, the
usually higher density of microbes in the soil environment
encourages genetic exchanges, which could enhance the
development of microbial resistance in the presence of
antibiotics (Murray 1997).
Effects of antibiotics on soil microbial community structure
Microbial community structure can change upon exposure
to antibiotics. The inherent reason is that antibiotics in
general, even those designed to be broad-spectrum drugs,
have their selective effects on various groups of microbes.
Here, the group of microbes could be as broad as fungi or
bacteria (Mohamed et al. 2005), and as narrow as a single
genus (Yang et al. 2009) or species (Moenne-Loccoz et al.
2001). As a result, the selective antibiotic effects alter the
relative abundance of microbial species, and subsequently
interfere with the interactions among different species.
Table 2 gives a comprehensive summary of studies on
antibiotic effects on microbial communities.
A number of studies were conducted to investigate the
phylogenetic structures of soil microbial communities
under antibiotic pressure by molecular fingerprinting
techniques, such as denaturing gradient gel electrophoresis
Table 2 Antibiotic effects on microbial community in natural environment

Antibiotic Concentrationa Parameter tested Effectb Unaffected Experimental Time length Detection method Reference
mg/kg parameter condition

Amoxicillin 10∼100 Resistance Increased Resistance Soil (applied with 18 days CFU count, Binh et al. 2007
transferability transferability in manure before) Exogenous isolation
(number of soil that was never amended with of plasmids, DGGE
transconjugants with applied with manure
amoxicillin resistance) manure before
Bacteria DGGE profile Altered
Ciprofloxacin 0.43∼5 mg/L Colonization resistance Decreased, Colonization Chemostat inoculated 41 days CFU count in agar Carman et al.
influent to Salmonella especially for 0.43 resistance at from human fecal plates 2004
kedougou mg/L 0.1 mg/L influent microflora, with
Salmonella in
Appl Microbiol Biotechnol (2010) 87:925–941

influent
0.2∼2 mg/L Pyrene mineralization Decreased Marine sediment with 11 weeks Radiorespirometry Naslund et al.
rate significantly ciprofloxacin in the measuring CO2 2008
overlying water production
Microbial community Altered significantly 7 weeks T-RFLP
0.2∼200 mg/L Biomass, PLFA Increased with Salt marsh sediment 30 days PLFA Córdova-Kreylos
richness, sulfate ciprofloxacin with ciprofloxacin in and
reducer and Gram- concentrations the overlying water Scow 2007
negative bacteria
markers
Indicators of Decreased
starvation stress:
17 cy/precursor
and saturated/
unsaturated
biomarker ratios
Lincomycin 0.05∼500 T-RFLP profile (16S Altered significantly Percentage of Soils with both high Not mentioned CFU count, T-RFLP, Čermák et al.
rRNA genes) antibiotic and low pH (>10 days) 2008
T-RFLP profile Altered significantly producers in soil
(lincomycin resistance with lower pH,
gene homologues) numbers of
Percentage of Decreased cultivated bacteria
actinomycetes among significantly and their resistance
total microbes in both soils
Percentage of antibiotic Decreased Soil with higher pH
producers among significantly
actinomycetes
Natamycin 50∼200 mg/L Bacterial community Altered significantly Bulk soil and 7 days Culturable cell Mohamed et al.
structure (from rhizosphere soil counting/bacterial 2005
ribosomal intergenic suspensions on agar community
spacer analysis) medium fingerprinting by
Ratio of fungi/bacteria Decreased ribosomal intergenic
CFUs significantly spacer analysis
927
Table 2 (continued)
928

Antibiotic Concentrationa Parameter tested Effectb Unaffected Experimental Time length Detection method Reference
mg/kg parameter condition

Oxolinic acid 9 g/kg of feed Microbial community Unique strains were Water and sediment in Not mentioned Bacteria isolation and Tendencia and
given once a isolated only in shrimp pond sensitivity test de la Pena 2001
week in shrimp pond with oxolinic
pond acid feed
Percentage of antibiotic- Highest in pond
resistant isolates with oxolinic acid
feed
Oxytetracycline 10 Bacteria/Actinomyces Decreased by Fungi CFUs, acidic Wheat rhizosphere 5∼30 days CFU count in agar Yang et al. 2009
CFUs 22.2%/31.7%, phosphatase/ soil plates, enzyme
respectively dehydrogenase/ activity assay
Alkaline phosphatase Decreased by 41.3% urease activity
activity
>20 µg/L Selection rate of Increased Mesocosms 56 days Quantitative real time Knapp et al. 2008
resistance gene containingpristine PCR (qPCR)
250 µg/L Ratio of total Increased surface water
tetracycline resistance
gene to 16s rRNA
0.81∼0.93 Substrate-induced Decreased by 10% Soil basal Pristine topsoil 24∼48 h SIR/iron(III) reduction Thiele-Bruhn and
respiration respiration/ amended with activity assay Beck 2005
5.5∼7.35 Iron(III) reduction Decreased by 10% dehydrogenase milled maize straw 7 days
activity activity or glucose
100∼1000 Ratio of fungi/bacteria Increased 14 days Fumigation-extracted
significantly microbial C and
ergosterol
concentration
Streptomycin 400 mg/L Ammonia oxidation Inhibited by 75% Activated sludge 2∼4 h Nitrifying activity Tomlinso et al.
calculated from 1966
nitrite and nitrate
production
Sulfachloropyridazine 115 PICT Increased Soil amended with 7 days PICT Schmitt et
significantly fresh pig slurry al. 2005
100 PICT Soil amended with
alfalfa meal
Sulfadiazine 1 PICT Increased by a factor Hotspot soil 5∼15 weeks PICT Brandt et al. 2009
of 2∼5
10∼100 Increased by a factor Bulk soil
of 1.5∼3.5
10∼100 Ratio of AOA/AOB Increase notably Soil amended with 61 days qPCR targeting AOA/ Schauss et al.
pig manure AOB ammonia- 2009
oxidizing genes
10∼100 Ratio of fungi/bacteria Increased in some G−/G+bacteria ratio Soil amended with 61 days PLFA Hammesfahr et al.
PLFA cases and Shannon– pig manure 2008
Bacteria/β- Eight/three/five Wiener index 32 days DGGE
Appl Microbiol Biotechnol (2010) 87:925–941
Table 2 (continued)

Antibiotic Concentrationa Parameter tested Effectb Unaffected Experimental Time length Detection method Reference
mg/kg parameter condition

proteobacteria/ bands changed, based on PLFA

pseudomonads DGGE respectively
profiles
10∼100 Sulfadiazine-resistant Increased Silt loam and a loamy 32∼61 days Culturable bacteria Heuer and Smalla
bacteria counts significantly sand mixed with counting (MPNs) 2007
Sulfadiazine resistance Increased manure containing qPCR
gene (sul1) frequency significantly sulfadiazine
10 Ammonia oxidization Decreased Soil basal Soil amended with 32 days Microbial activity Kotzerke et al.
activity respiration manure assay 2008
100 Soil basal respiration Decreased
Ammonia oxidization Decreased
activity
Denitrification Decreased slightly
1∼50 Bacteria DGGE profile Shifts in band Bacteria DGGE Soil amended with 48 days DGGE Zielezny et al.
intensity profile/soil basal glucose 2006
Appl Microbiol Biotechnol (2010) 87:925–941

Diversity (Shannon– Decreased at first respiration in

Wiener index) then recovered unamended soil
after 48 days
Soil basal respiration Decreased Soil respiratory
significantly activity assay
>0.15 (introduced Sulfadiazine resistance Influenced Soil amended with 175 days qPCR Heuer et al. 2008
by sulfadiazine gene (sul2) frequency significantly manure containing
residue in sulfadiazine
manure)
1∼100 Ratio of sul2 to 16 S Increased by Soil amended with 61 days qPCR Heuer et al. 2009
rRNA gene copies 1∼2 log units manure
Sulfamethoxazole 20∼500 PICT Increased by a factor PICT when Soil amended with 1∼5 weeks PLFA/PICT Demoling et al.
of 2 antibiotic is <1 manure from alfalfa 2009
Ratio of fungi/bacteria Increased or antibiotic-treated
PLFA pigs
500 PICT Increased by a factor Unamended soil
of 2
Sulfapyridine 0.05∼1.17 Substrate-induced Decreased by 10% Soil basal Pristine topsoil 24∼48 h SIR/iron(III) reduction Thiele-Bruhn and
respiration respiration/ amended with assay Beck 2005
0.003∼1.14 Iron(III) reduction Decreased by 10% dehydrogenase milled maize straw 7 days
activity activity or glucose
100∼1000 Ratio of fungi/bacteria Increased 14 days Fumigation-extracted
significantly microbial C and
ergosterol
concentration
Tetracycline 5∼500 Ratio of G+/G− PLFA Decreased Tetracycline Soil with or without 8 weeks PLFA, qPCR Hund-Rinke et al.
Ratio of fungi/bacteria Increased resistance gene pig manure (rich in 2004
PLFA copies tetracycline
resistance gene)
0.003∼0.1 Tetracycline-resistant CFUs increased Soil spread with pig 8 months CFU count Sengeløv et al.
bacteria after spread of manure containing 2003
manure but tetracycline
declined to control
929
Table 2 (continued)
930

Antibiotic Concentrationa Parameter tested Effectb Unaffected Experimental Time length Detection method Reference
mg/kg parameter condition

level later
Tylosin 2000 Diversity of microbial Decreased as seen Fungi growth Sandy soil 2 months Colony morphology, Westergaard et al.
community (number on days 15 and 22, DGGE, sole carbon 2001
of bands on DGGE but return to source utilization
profiles) control level on
day 33
Microbial community Altered
structure (DGGE band
patterns)
Percentage of tylosin Increased from less
resistant CFUs than 1% to above
30%
2000 Substrate-induced Increased Diversity of Soil amended with 60 days Colony morphology, Müller et al. 2002
respiration microbial substrates DGGE, sole carbon
community source utilization
50∼1500 PICT Initially increased, Soil with or without 95 days PICT Demoling and
then decreased alfalfa Bååth 2008
after 25 days, and
return to control
level after 95 days
>50 Bacteria growth rate Decreased, with 2 days
IC50 of 960 mg/kg
soil

a
If not indicated otherwise
b
Compared with control treatment without antibiotic, if not indicated otherwise
Appl Microbiol Biotechnol (2010) 87:925–941
Appl Microbiol Biotechnol (2010) 87:925–941
(DGGE) and terminal restriction fragment length polymor-
phism (T-RFLP). Antibiotics studied include amoxicillin
(Binh et al. 2007), lincomycin (Čermák et al. 2008),
natamycin (Mohamed et al. 2005), and sulfadiazine
(Zielezny et al. 2006; Hammesfahr et al. 2008). Most often,
after treatment of soil with antibiotics, microbial commu-
nity fingerprinting techniques revealed emergence and
disappearance of signals representing different microbial
species. Even though some cases showed identical band
patterns among treatments, intensities of the signals still
varied significantly (Hammesfahr et al. 2008). Notably,
effects on microbial population are dependent on the
original soil properties (Čermák et al. 2008), microbial
groups (Hammesfahr et al. 2008), and the dose of anti-
biotics added (Zielezny et al. 2006). In Hammesfahr et al.'s
(2008) study on sulfadiazine effects, changes in DGGE
profiles of pseudomonads and β-proteobacteria were less
pronounced than bacteria, possibly due to the intrinsic
resistance of the strains in these two groups. Additionally,
another report showed that 10∼50 mg/kg sulfadiazine
exerted much stronger influence on DGGE band pattern
intensities, though 1 mg/kg sulfadiazine only evoked
minimal effects on soil communities (Zielezny et al. 2006).
The above-mentioned studies suggest that the commu-
nity composition changes caused by antibiotics often last
throughout the experimental periods. However, as a general
community parameter, diversity of the community may
recover during the antibiotic incubation after as short as
15 days, even though the composition of the community
remains altered (Westergaard et al. 2001). Similarly, another
study showed that the diversity of soil decreased signifi-
cantly at the beginning of the experiment with 1∼10 mg/kg
sulfadiazine, however, later the community diversity recov-
ered to the same level as the control soil after 48-day
incubation (Zielezny et al. 2006). This phenomenon reflects
the soil's ability to recover from antibiotic disturbance, and
is important since the diversity is highly correlated with the
ecological functions of soil (Müller et al. 2002).
Due to the complexity of signals acquired from the
community fingerprinting techniques, statistical analysis
(e.g., cluster analysis or principal component analysis) is
often applied to show the differences of microbial commu-
nity at various stages. However, no detailed information on
the affected species can be retrieved from the results, unless
specific bands are sequenced after DGGE analysis, which is
both time-consuming and inaccurate due to the limitation of
DGGE (Müller et al. 2002). As such, DNA microarray
might be better suited to investigate phylogenetic changes
under antibiotic pressure (Fan et al. 2009).
Additionally, the ratios of bacteria/fungi and Gram-
positive/Gram-negative bacteria (G+/G−) are two most
often used indicators to tell changes in microbial commu-
nity structure. Many studies have been performed on the
931
fungi/bacteria ratio in soil upon exposure to various
antibiotics (Thiele-Bruhn 2005; Hammesfahr et al. 2008;
Demoling et al. 2009; Yang et al. 2009), since the ratio is
tightly linked with the function of soil ecosystem (Poly-
anskaya et al. 1995; Bailey et al. 2002; Bååth and Anderson
2003). The antibacterial sulfadiazine (at concentrations of
10∼100 mg/kg) increased the fungi/bacteria ratio in
manured soils, due to both a reduction in bacteria biomass
and a stimulated growth of fungi cells at the beginning of
incubation (Hammesfahr et al. 2008). However, the effect
of antibiotic is not as significant as an application of
manure, which increased the fungi/bacteria ratio by at least
40% compared with the control soil. Other antibacterials,
such as sulfapyridine/oxytetracycline (Thiele-Bruhn and
Beck 2005), and sulfamethoxazole (Demoling et al. 2009)
could also increase fungi/bacteria ratio as expected. It is
noteworthy that antimicrobial agents may act on non-target
organisms as a result of the indistinct boundary of their
effective groups. Antibacterial oxytetracycline and penicil-
lin, when combined together, reduced the lengths of active
hyphae of fungi in forest soils (Colinas et al. 1994).
Meanwhile, fungicides like natamycin and captan also exert
antibacterial effects. Natamycin, which is commonly used
to control fungal growth on agar media, showed significant
inhibition on bacteria, both in cell numbers and community
diversity, although the effective concentration is higher than
that for fungus (200 versus 50∼100 mg/L) (Mohamed et al.
2005). Captan, as a fungicide, reduced more than 50% of
active bacteria at 25 mg/kg (Colinas et al. 1994) and
inhibited both bacteria and fungi at 400 mg/kg (Lin and
Brookes 1999). The bactericide bronopol, while exhibiting
effects in increasing fungi/bacteria ratio, also showed
antifungal activity in soil at a concentration of 200 mg/kg
(Lin and Brookes 1999). The antibacterial tylosin has no
apparent effects on soil fungi population, but rather
indirectly influences their biomass by changing bacterial
populations. In tylosin-treated soils, fungi biomass was
found to increase continuously during the first 2 weeks,
possibly due to competition displacement since bacterial
colony forming units were greatly reduced during this
period. The amplification of fungi biomass might also apply
to other antibacterial agents that exert strong suppression on
bacteria while having rather small effects on fungi. Given
that agricultural plants are often susceptible to pathogenic
fungi (Rossman 2009), the increase in fungi abundance in
soil may threaten the yield and quality of agriculture
products. Although antibiotics with antifungal activity have
been extensively studied on their effects on plant diseases
caused by fungi in agriculture (Worthington 1988), little is
known about the impact of antibiotic contamination on the
possible occurrence of plant diseases in the agricultural soil.
G+and G− bacteria react differently to quite a number of
antibiotics due to their distinct cell wall structure (Table 2).
932
For example, the broadband antibiotic sulfadiazine did not
affect G+/G− ratio significantly (Hammesfahr et al. 2008),
while tetracycline decreased the G+/G− ratio because the
bacteria resistant to tetracycline are more likely to be Gram-
negative (Schnabel and Jones 1999; Hund-Rinke et al.
2004).
Effects of antibiotics on antimicrobial resistance in soil
Development of antibiotic-resistant microbes as well as
resistance genes are attracting increasing attention because
of their impending influence on the effectiveness of
clinically applied antibiotics. It is crucial to study the
casual linkage between the release of antibiotics and
proliferation of antibiotic resistance in order to provide a
basis for decision makers in controlling the usage of
antibiotics (Ghosh and LaPara 2007).
The changes in community tolerance after exposure to a
certain pollutant can be measured by pollution-induced
community tolerance (PICT) (Blanck 2002; Boivin et al.
2002). PICT changes under antibiotic treatment not only
reflect shifts in microbial community structure, but also
imply the amplification of antibiotic resistance within the
community. Sulfonamides are one group of the frequent
targets of PICT studies in soil because of their wide usage
in livestock (Thiele-Bruhn 2003) and their persistence in
liquid manures and soil (Sukul and Spiteller 2006). Several
studies found substantial PICT increase after the soil was
treated with sulfonamides, e.g., sulfadiazine (Brandt et al.
2009), sulfamethoxazole (Demoling et al. 2009) or sulfa-
chloropyridazine (Schmitt et al. 2005). In addition to the
sulfonamide class, another bacteriostatic antibiotic—tylosin
belonging to the macrolide class, could also invoke
temporary PICT increase in tylosin-polluted soil (Demoling
and Bååth 2008), however, PICT values returned to pre-
pollution levels after tylosin disappeared in the soil. Rather
than selection for resistant microbes, the increase of PICT
may also be due to the adaptation of existing organisms.
Therefore, PICT is only an indirect way to study bacteria
resistance, and it is often combined with other methods (e.
g., phospholipid fatty acid analysis (PLFA)) to indicate
concurrent structural changes in microbial communities.
Increases in resistant microbes and resistance genes are
more direct evidence for the expansion of resistance, and
they have been widely observed in both soil and water
environment (Kümmerer 2004; Patterson et al. 2007; Malik
et al. 2008; Zhang et al. 2009). In a study of archived soil
spanning from 1940 to 2008 in the Netherlands, all 18
tested antibiotic resistance genes have significantly in-
creased since 1940, especially tetracycline resistance genes
(Knapp et al. 2010). Ghosh and LaPara (2007) suggested
that the use of chlortetracycline at sub-therapeutic concen-
trations together with excessive use of animal manure
Appl Microbiol Biotechnol (2010) 87:925–941
facilitated the spread of bacteria resistance, probably due to
horizontal gene transfer. Heuer and Smalla (2007) also
showed that manure from antibiotic-free sources plus
sulfadiazine synergistically increased sulfadiazine-resistant
bacteria count in soil after 2 months. The same research
group later found that the abundance of sulfadiazine
resistance gene copies also increased during the same
incubation period (Heuer et al. 2009). They inferred that
horizontal gene transfer might have occurred between
manure bacteria and indigenous soil bacteria, resulting in
an increase of resistance in soil.
In an attempt to elucidate the dynamics of sulfonamide
resistance genes in manured soil, Heuer et al. (2008)
modified the mathematic model by including the positive
selection of resistance gene caused by antibiotics, apart
from the cost of sulfadiazine resistance genes and horizon-
tal gene transfer. The modeling agrees well with the
frequency of developed resistance gene copies with various
concentrations of sulfadiazine, and shows a selective effect
of sulfadiazine at 0.15 mg/kg.
In contrast, in some other studies, antibiotics do not
seem to pose significant effects on the amplification of
resistance in soil microbes, especially when applied with
manure. In a report of Sengeløv et al. (2003), pig manure
slurry containing residual antibiotics (primarily tetracy-
clines and lincomycin) was spread onto soil to mimic
disposal of manure onto soil surface. Although the
resistance among soil bacteria increased temporarily, it
returned to preapplication levels within 5 months. The
temporary increase in resistant bacteria was attributed to the
input of resistant intestinal bacteria with limited survival
potential in the soil environment. Similarly, in another
study aiming at tracking resistance gene, the addition of
tetracycline to the samples had no effect on the establish-
ment of resistance genes (Hund-Rinke et al. 2004). During
the incubation period, the amount of resistance genes even
decreased, possibly because the initial high amount of
resistance genes was introduced by the application of
manure rich in tetracycline-resistant genes. These exotic
resistance genes could have masked the effect of antibiotics
on resistance which existed but were not adequate to show
up in the limited testing period. It is not known yet whether
the discrepancy in the results of Sengeløv et al. (2003) and
Heuer et al. (2009) is due to differences in mobility of
tetracycline and sulfadiazine resistance genes or differences
in the incubation time (365 versus 61 days). After all, the
contribution of horizontal gene transfer and the presence of
antibiotics to resistance expansion in soil after manure
application still need further study.
In all, resistance expansion in the natural environment is
a complex process controlled by multiple factors, while
antibiotic input is only one of the many reasons for the
resistance development. Example from the swine industry
Appl Microbiol Biotechnol (2010) 87:925–941
indicates that even a total ban of antibiotics could not solve
the problem of resistance expansion and the antibiotic
resistance in fecal coliform still exists (Cromwell 2002). In
the same study, researchers found that factors such as pig
age, housing system, and moving stress had as much or
even more effect on resistance than the presence of
antibiotics in the diet.
Effects of antibiotics on ecological functions of soil
As early as in the 1980s, Macri et al. (1988) has predicted
the potential impacts of antibiotics on ecological services of
microbes. At similar time, Fedler and Day (1985) found
that chlortetracycline (18 mg/L) inhibited methane produc-
tion under mesophilic conditions. However, till now,
knowledge in this field is still limited. Two studies on
sulfadiazine (Kotzerke et al. 2008; Schauss et al. 2009) are
currently the only set of investigations on antibiotic effects
on soil nitrification/denitrification processes. Sulfadiazine
at 10∼100 mg/kg reduced manure's growth stimulation
effect on ammonia-oxidizing bacteria (AOB) notably, while
ammonia-oxidizing archaea (AOA) was affected to a lesser
extent and even exhibited growth with sulfadiazine at
100 mg/kg (Schauss et al. 2009). The observed stronger
resistance of AOA than AOB towards sulfadiazine might be
due to the differences in archaeal and bacterial cell walls
and membranes. In a soil sample with higher AOA/AOB
ratio (73:1), ammonia oxidation recovered during days 32
to 61. While in the other soil sample with an AOA/AOB
ratio of 7:1, this activity was inhibited throughout the
experiment. By model calculation, the authors evaluated
the contribution of AOA activity to ammonia oxidation in
the soil with higher AOA/AOB ratio. Results showed that
AOAs contribution increased from 50% to 70% under the
sulfadiazine stress, indicating the functional redundancy
between AOA and AOB. In all, the soil ecosystem seems to
have a mechanism to buffer the antibiotic stress and recover
from the inhibition of certain sensitive microbes by
substituting their functions with less sensitive but function-
ally redundant groups. Apart from the nitrification process,
Kotzerke et al. (2008) also studied sulfadiazine’s effects on
denitrification process in soil. With sulfadiazine at 10–
100 mg/kg, denitrification rate in both soil samples
(manured or non-manured before) was significantly re-
duced, and the effects were measureable after only 1-day
exposure.
Previous studies also reported antibiotics effects on
microbial activities and enzymes in soil. Soil biomass
production and soil respiration rate could be affected by
various antibiotics, such as ciprofloxacin (Naslund et al.
2008), sulfadiazine (Zielezny et al. 2006; Kotzerke
et al. 2008) and tylosin (Müller et al. 2002; Demoling and
Bååth 2008). The soil enzyme alkaline phosphatase
933
declined from 41.3% to 80.8% with 10 ∼ 30 mg/kg
oxytetracycline in soil, while the other three soil enzymes
tested (acidic phosphatase, dehydrogenase, and urease)
were not affected (Yang et al. 2009). Thiele-Bruhn and
Beck (2005) reported that basal respiration and dehydroge-
nase activity in soil were not significantly affected by either
sulfapyridine or oxytetracycline regardless of the dosages
of antibiotics. The authors explained the results in the
following possible reasons: (1) a low sensitivity of the
detection methods for the two soil parameters; (2) selective
effects of the antibiotics that were not indicated by the
microbial activities measured with the two methods; (3)
shifts in the microbial community structure that compen-
sated for effects on single species; (4) the bacteriostatic but
not bactericidal properties of sulfapyridine and oxytetracy-
cline, which means that they inhibit the bacteria growth but
not kill them. In another study (Thiele-Bruhn 2005), dose-
related inhibition of microbial iron(III) reduction were
found in seven out of nine antibiotics investigated (three
sulfonamides, five tetracyclines, and benzimidazole). So
far, studies on microbial activity parameters in soil are
restricted in the group of bacteriostatic antibiotics that often
require additional substrate to magnify their effects, while
bactericidal antibiotics are still vague in their effects in soil
microbial functions.
In summary, previous studies show that antibiotics affect
microbial ecological functions. From the existing results,
soil ecosystems show their resilience and may alleviate the
effects of antibiotics with the help of redundant functional
groups and recovery of resistant microbes. However, most
investigations have been conducted on bacteriostatic anti-
biotics, and soil services other than nitrogen transformation
have not been touched much.
Effects of antibiotics on aquatic microbial community
Despite of the vast extent of aquatic ecosystems and the
frequent occurrence of antibiotics in surface waters
(Watkinson et al. 2009), studies on effects of antibiotics
in the water body are less compared to studies on soil
environment. This is probably due to the following reasons.
1. Antibiotic effects in water are less pronounced than
those in soil because of relatively lower concentra-
tions, probably caused by dilutions (especially in
dynamic water bodies), aerobic/anaerobic biotrans-
formation, and adsorption by sediment or biofilm.
The values detected in the aquatic environment are
often in the nanogram and lower microgram per liter
range (Kümmerer 2009b), while the values could go
up to milligram per kilogram range in soil (Thiele-
Bruhn 2003).
934
2. Aquatic microbes are often not localized, and are
present in much lower density than those in soil,
adding to the difficulty in detection and comparison of
microbial community structure.
3. Studies on resistance development often focus on the
influence of resistant bacteria and resistance gene brought
by wastewater rather than on the antibiotics present in the
water body, since effluent from sewage treatment plant is
one of the major sources of resistance and antibiotics in
the aquatic ecosystems (Kümmerer 2009a).
However, the study of antibiotic effects in aquatic environ-
ment is of significance. Less pronounced effects at lower
concentration and shorter exposure time do not necessarily
mean weaker impacts on the aquatic ecosystems than on the
soil environment. Compared with soil, the water environment
has a more direct and intimate contact with human life due to
the wide usage of water and the continuity of the water body
that may transport antibiotic/resistance to every corner of the
world, posing threat to public health and ecosystems. Also,
evidence has shown that even in sub-inhibitory level concen-
trations, antibiotics may still exert their impact on microbial
community (e.g., by influencing transcription in microbes)
(Davies et al. 2006), and long-term effects of exposure to low
concentrations of antibiotics are still largely unknown.
Currently, limited information is available on whether the
presence of antibiotics in the natural habitats could cause the
expansion of resistance in water body (Kümmerer 2009a),
though antibiotic-resistant bacteria and their resistance genes
are often detected in aquatic ecosystems as well as
wastewater and drinking water (Esiobu et al. 2002; Schwartz
et al. 2003; Biyela et al. 2004). Tendencia and de la Pena
(2001) found that antibiotic resistance was associated with
historical and current use of antimicrobial, which was found
to be highest in the shrimp pond currently using oxolinic
acid in feed. Another study showed an increase in
tetracycline resistance genes abundance (compared to 16S
rRNA genes) at low-level oxytetracycline exposure in
aquatic systems (Knapp et al. 2008). Therefore, antibiotic
input deserves equal attention as resistance input for their
effects on resistance amplification in aquatic environment.
The changes in ecological functions of aquatic environ-
ment caused by antibiotics have been investigated in several
studies. Nitrogen transformation process has been studied
since 1960s (Tomlinso et al. 1966), in which streptomycin at
400 mg/L was required to achieve 75% inhibition of
ammonia oxidation in activated sludge in 2∼4 h. In contrast,
ampicillin (0–250 mg/L), benzylpenicillin (0–250 mg/L),
novobiocin (0–150 mg/L), oxytetracycline (0–250 mg/L),
and chloramphenicol (0–50 mg/L) showed no effect on
either biomass or nitrate production in a stabilized nitrifying
sludge within 80 h (Gomez et al. 1996). It has also been
shown that antibiotics such as oxytetracycline inhibit
Appl Microbiol Biotechnol (2010) 87:925–941
nitrification process in surface water (Klaver and Matthews
1994). However, in some cases, when a complex mixture of
bacteria is exposed to antibiotics, increased nitrification
activity can be observed, the reason of which is not yet
clear (Halling-Sørensen 2001; Alexy et al. 2001). In another
interesting study conducted on sequencing batch reactors
(SBRs), although no apparent changes were observed for
nitrogen or phosphorus removal upon continuous exposure
to trace amount of erythromycin (100 µg/L), microbial
community structures shifted significantly as analyzed by
high-density microarrays (Phylo-Chip) (Fan et al. 2009).
This implies that the functional redundancy within the SBR
system may make up for the loss of sensitive bacteria under
antibiotic pressure. Moreover, methanogenesis has been
reported to respond to antibiotics such as sulfamethoxazole
and ofloxacin, which caused a mild inhibition to methano-
gens in an anaerobic digestion process (Fountoulakis et al.
2004). Ofloxacin was further studied on its extent and type
of impact on the kinetics of methanogenesis and aceto-
genesis (the two key steps in anaerobic digestion), and once
more, mild inhibition to both processes were found
(Fountoulakis et al. 2008).
Primarily due to aquaculture usage, antibiotics in sediments
sometimes could raise up to several hundred milligrams per
kilogram (Thiele-Bruhn 2003). As a result, antibiotic effects
are found in sediment overlay water environment, such as
reduction of bacteria numbers, resistance expansion and
effects on ecological functions. Based on PLFA analysis,
ciprofloxacin, as a broad-spectrum antibiotic, favored sulfate-
reducing bacteria and Gram-negative bacteria in salt marsh
sediment (Córdova-Kreylos and Scow 2007), while it
affected both the diversity of bacteria community and the
degradation of the PAH pyrene in another study (Naslund et
al. 2008). These two cases showed consistency in discovering
similar ciprofloxacin inhibition concentration (∼200 µg/L) in
the sediment overlay water. However, sulfate reduction rate
was found to reduce to 10% of that in control tank 7 days
after oxytetracycline, oxolinic acid, or flumequine was added
into the artificial marine fish farm sediment, and the effects
lasted for at least 29 days (Hansen et al. 1992).
Factors that influence antibiotic effects
Both the inherent chemical properties of antibiotics and the
environmental parameters could influence the actual effects
of antibiotics on microbial community. A deeper under-
standing of these factors will add to our knowledge on how
to predict and control these antibiotic effects.
Concentration
The fate of antibiotics in soil and aquatic sediments mainly
depends on sorption, transportation, and transformation
Appl Microbiol Biotechnol (2010) 87:925–941
(Tolls 2001; Thiele-Bruhn 2003). Accordingly, the effects
of antibiotics are influenced by their actual concentrations
in soil that could get direct contact with microbes
(bioavailable concentration). The actual concentration in
soil and aqueous phase is a function of time, stability or
persistence of the antibiotic, soil properties (such as soil
pH, soil organic matter, and soil minerals), and microbial
activity (Thiele-Bruhn 2003). Sorption of antibiotics in soil
is tightly related to soil pH (Gu and Karthikeyan 2005;
Basakcilardan-Kabakci et al. 2007). Also, environmental
pH influences the charge of antibiotics presented in soil or
water (Kümmerer 2009a), which in turn affects their
stability (Skinner et al. 1993; Aksu and Tunc 2005). Both
effects (sorption and charge of antibiotics) determine the
bioavailable concentration of antibiotics. In the study of
Čermák et al. (2008), two forest soils differing in pH,
humic acid and clay content were investigated for linco-
mycin effects without addition of any substrate. Usually,
soil with higher clay content possesses higher sorption
ability towards antibiotics, resulting in lower antibiotic
effects (Figueroa et al. 2004; Hu and Coats 2009).
However, results showed that the bacterial community of the
high-clay-content soil was more affected by lincomycin than
the other soil with lower pH and higher content of humic
acids. Therefore, pH and humic acid content could play a
more important role in diminishing the antibiotic effect (e.g.,
by inactivation of antibiotics). This is supported by the studies
of Wang et al. (2009) and Gu and Karthikeyan (2008), which
addressed the importance of pH and humic substance on the
mobility of antibiotics in soil, respectively. In the study of
Wang et al., pH-dependent sorption was observed for
lincomycin, and as the solution pH increased, lincomycin
sorption decreased, corresponding well with the results of
Čermák et al. In another study carried out by Tappe et al.
(2008), the relationship between pH and susceptibility of
bacteria towards sulfonamides fits well with model predic-
tion, a strong support for the significance of pH in the
antibiotic effects on microorganisms. Moreover, a number of
studies showed that sorption may lead to the reduction
(Thiele-Bruhn 2005; Córdova-Kreylos and Scow 2007;
Kotzerke et al. 2008) or even disappearance (Hund-Rinke
et al. 2004) of antibiotic effects on microbial communities.
Most of the antibiotics studies exhibit a dose-dependent
relationship with their effects on microbial communities
(Mohamed et al. 2005; Naslund et al. 2008; Brandt et al.
2009; Demoling et al. 2009). Halling-Sørensen et al. (1998)
came up with a comprehensive table listing effective/lethal
concentrations of different antibiotics and their target
organisms, based on acute toxicity test. However, many of
the tested concentrations of antibiotics are unlikely to be
found in the environment. As an exception, in a river water
body near the effluent of sewage treatment plant (Larsson et
al. 2007) or manured soils (Demoling et al. 2009) where
935
dilution effects are not significant, antibiotic concentrations
may go up to a level comparable to those in the experiments.
On the other hand, even at low concentrations, some studies
still suggest antibiotic effects on microbes. This is supported
by the observation that sulfapyridine and oxytetracycline
exert a temporary selective pressure on soil microorganisms
even at environmentally relevant concentrations (0.003–
7.35 mg/kg) (Thiele-Bruhn and Beck 2005). Furthermore,
certain gene transcriptions which are not necessarily associ-
ated with stress responses could also be affected by
structurally diverse antibiotics in sub-inhibitory concentra-
tions (Davies et al. 2006). In all, antibiotics may play roles
other than as growth inhibitors, especially in the low
concentration levels mentioned above (Little et al. 2008).
Exposure time
It is natural to perceive that exposure time can be prolonged in
order to allow a latent effect to become detectable. Thiele-
Bruhn (2003) summarized the inhibition rate and time
required for various antibiotics at specific concentrations
towards organisms in the habitat. In two soil samples (sandy
soil and loamy soil), Thiele-Bruhn and Beck (2005) initially
tried to detect substrate-induced respiration (SIR) change in
soil within 4 h by applying sulfapyridine or oxytetracycline,
but the SIR was not influenced even at a concentration of
1,000 mg/kg. Subsequently, the incubation time was
extended to 24 and 48 h, respectively. Strong inhibition
effects were observed in a clear dose-related manner for the
sandy soil after 24-h incubation, while these were only seen
after 48-h incubation for the loamy soil. Both results suggest
that short-term tests may be not suitable for bacteriostatic
antibiotics such as sulfonamide and tetracycline.
However, long-term studies frequently detect recovery of
the community biomass and return of certain microbes that
were initially inhibited. Demoling et al. (2009) employed
PLFA and PICT analysis to evaluate the effects of
sulfamethoxazole on soil microbes, which demonstrated
an initial decrease in bacterial growth rates and a gradual
amplification of more tolerant species as reflected by PICT
increase. Similarly, in Kotzerke et al. (2008), microbial
activity recovered at day 32 when high concentration of
sulfadiazine was fed. The recovery ability of the microbes
against antibiotics was also observed on specific microbes
rather than on whole community scale. Return of specific
microorganisms after antibiotic treatment was also observed
in mouse gut microbiota. Using PCR-DGGE technique,
Yap et al. (2008) found that sequences closely related to
Clostridium leptum and Clostridium coccoides from mouse
intestine initially disappeared from the DGGE profiles at
day 1 but recovered at days 2 and 3 post-treatment with
vancomycin. This study of antibiotic effect on mammal
microflora sheds light on the effects of antibiotic on
936
environmental microbes, suggesting that not only does the
overall function of microbial communities tend to recover
after antibiotic treatment, specific groups may also reappear
in the microbial profiles. Nevertheless, Westergaard et al.
(2001) found that the diversity of soil microbial community
returned to control level at day 15 after treatment by
tylosin, but the community composition remained changed.
The resilience and robustness of the micro-ecosystem under
antibiotic pressure continue to be an interesting topic, and it
is still under debate whether diversity contributes to the
stability of the system (Little et al. 2008).
Till date, all of the studies regarding antibiotic effect on
natural habitats are based on a single application of
antibiotics. However, it is an over-simplification of the real
environment, where the input of antibiotics could be in a
single pulse, multiple pulses, or even in a continuous mode.
It would be interesting to see how the measured parameters
differ if the antibiotics are applied in various modes. It is
possible that an already adapted microbial population will
not be influenced by other antibiotics after several
applications. Another suggestion is keeping the antibiotics
at a constant level throughout the experiment. This constant
but low level of antibiotics may cause unexpected and
prominent effects in the community structure of soil or
aquatic environment. This approach could be applied to
study the long-term effects of low antibiotic concentration,
the impact of which is latent in the short-term treatment.
However, it is worth to point out that some detection
methods on microbial community change (e.g., PICT) may
not be favorable for long-term incubation experiments
(Demoling et al. 2009). This is because PICT measures
the status of community tolerance at the time point of
sampling, and ideally no exposure towards the pollutants
should be imposed during the detection period in order to
avoid further increase of community tolerance.
An interesting issue regarding antibiotics exposure time
is that, as is known, short-term exposure causes significant
changes in community structure (especially in the presence
of substrate), but it is crucial to find out whether long-term
or even irreversible alteration to the microbial population
will take place after short-term exposure to antibiotics,
since antibiotics are often not persistent in natural habitat
due to adsorption or transformation. Clinical evidence has
shown that short-term antibiotic administration can cause
long-term alterations in microbiota (Lofmark et al. 2006).
In field studies, persistence or further increase of antibiotic
effects is often observed even after quick declination of
antibiotic amounts in soil, such as sulfonamides (Kotzerke
et al. 2008). However, studies also show that the effects on
microbial community tend to be attenuated and soil
parameters tend to rebound to normal level when the
antibiotic like tetracycline or tylosin disappear from soil
(Rysz and Alvarez 2004; Demoling and Bååth 2008).
Appl Microbiol Biotechnol (2010) 87:925–941
Hence, the persistency of antibiotic effects varies, possibly
according to the type of antibiotic used and also specific
testing environment. Such information needs to be collected
and summarized to provide a more accurate estimation on
the aftermath of antibiotic input into the environment.
Substrate
Apart from extended exposure time, adding substrate is
another way to amplify antibiotic effects, especially on
bacteriostatic antibiotics. Substrates such as glucose, root
exudate and manure slurry are reported to enhance
antibiotic effects on microbial community (de Oliveira et
al. 1995; Herron et al. 1998; Schmitt et al. 2005; Thiele-
Bruhn and Beck 2005; Zielezny et al. 2006). Noteworthy,
when using substrate to amplify effects of antibiotics, one
should bear in mind that: (1) substrate may distort the
microbial community structure because of stimulation of
specific microbial groups as is in rhizosphere (Perry et al.
1989); (2) root exudate may contain antibacterial substan-
ces (El-Mahmood and Doughari 2008; Jeyachandran et al.
2009; Puglisi et al. 2009), and also manure slurry resistance
has to be taken into consideration on disturbance of
antibiotic effects. Schmitt et al. (2005) and Heuer and
Smalla (2007) showed that manure stimulated PICT
increase in soil when treated with sulfachloropyridazine
and sulfadiazine, respectively. However, manure may bring
in resistant bacteria and resistance genes (Binh et al. 2008),
and may change soil properties as well. Therefore, without
further investigations, it would be difficult to separate the
effects of nutrients within manure and the introduced
resistance. Regarding this issue, Hammesfahr et al. (2008)
compared the DGGE pattern of pure manure with those of
the soil samples and found that manure populations did not
become major components of the soil community. Instead,
manure rather indirectly influenced soil microbial community
structure, e.g., by growth stimulation or genetic exchange as
discussed in the study of Heuer et al. (2008). Interestingly,
some antibiotics may even serve as carbon sources to some
of the bacteria (Dantas et al. 2008). This may explain
substantial increases in microbial biomass observed in salt
marsh sediments after adding ciprofloxacin as the only
carbon source (Córdova-Kreylos and Scow 2007).
The substrate effect resulted from plants is significant.
The high input of organic materials from plant roots and
root exudates (Vogt et al. 1982) not only promotes the
growth of microbes in the rhizosphere, but also modifies
the antibiotic impact to soil microbes. Mohamed et al.
(2005) demonstrated that natamycin concentrations varying
from 50 to 100 mg/L were sufficient to control fungal
growth in soil suspension from bulk soils. However, for the
rhizosphere soil, natamycin concentrations up to 200 mg/L
were required to control fungi growth. Using artificial root
Appl Microbiol Biotechnol (2010) 87:925–941
exudate as substrate amendment, Brandt et al. (2009) found
enhanced community tolerance at sulfadiazine of 1 mg/kg
in rhizosphere soil, while this effect was only observed at
sulfadiazine of 10∼100 mg/kg in bulk soil. Hence,
rhizosphere may be the key sites for proliferation of
resistant bacteria. However, as a biologically complex
microhabitat with the presence of both beneficial and
deleterious microorganisms as well as the plant hosts, it is
difficult to pinpoint the mechanisms behind this enhance-
ment of resistance. Antibacterial activity in plant root
exudates also has to be considered and tackled. In
summary, more studies are needed, especially on rhizo-
sphere of agricultural plants that are in proximity with
human habitat and are thus more probable to be exposed to
antibiotics.
Multiple antibiotics
There are a number of studies on the combined activity by
multiple antibiotics on bacteria strains under laboratory
conditions (Dutta et al. 2009; Ganiere and Denuault 2009;
Hames et al. 2009; Pietschmann et al. 2009; Tabak et al.
2009). Till now, there are few reports on the combined
effects of several antibiotics on microbial community in
field study, although environmental compartments like
surface water (Halling-Sørensen et al. 1998; Christian et
al. 2003; Liu et al. 2009), sewage (Liu et al. 2009;
Watkinson et al. 2009) and sediment (Bjorklund et al.
1991; Tang et al. 2009) have all been reported to harbor
multiple antibiotics simultaneously. Considering the various
types of combined effects of antibiotics (Keith et al. 2005;
Yeh et al. 2006), it is expected that the coexistence of two
or even more antibiotics in soil or aquatic environment will
present a different effect from when they are acting alone.
A study conducted by Christensen et al. (2006) is
currently the only case on effects of antibiotic combination
in environment. They observed synergistic effects with the
combination of erythromycin and oxytetracycline on
activated sludge microorganisms, while additive effects
were found with the combination of flumequine and
oxolinic acid. Surprisingly, combination of salinomycin
(antibiotic) and citric acid even changed the targeted group
of salinomycin by demonstrating bactericidal activity on
Gram-negative bacteria in addition to the conventional
target on Gram-positive bacteria (Ogita et al. 2009).
Another interesting phenomenon is that resistance to one
of the antibiotics could actually have the opposite effect in
suppressive multidrug treatments (Chait et al. 2007). In
such cases, although resistance would indeed diminish the
inhibition imposed by one of the antibiotics, it may also
remove the suppression among the antibiotics, rendering
the combined treatment more effective against the resistant
mutant than against the wild type. Therefore, a seemingly
937
paradoxical but rationalized phenomenon is observed, in
which under the suppressive antibiotic combination (doxy-
cycline–ciprofloxacin), a diminished selection for the
resistant mutant was found in comparison with doxycycline
alone. Thus, in particular cases, coexistence of more than
one antibiotic in the community is possible to even
decrease the overall resistance of the whole population
because of the removal of the suppression effect and
selection for the resistant group.
Summary
This review summarizes recent findings on the antibiotic
effects to microbial community in the context of natural
habitat. To assess the antibiotic effects in depth, important
parameters (e.g., concentration, exposure time, addition of
substrates) are also discussed. The following are important
conclusions and recommendations worth to be tackled in
the future study:
1. Phylogenetic analyses have revealed various changes
in microbial community structure caused by anti-
biotics; however, results are often explained statisti-
cally, offering no exact meaning for each change in
the community profiles. If those changes could be
located at finer levels (e.g., using microarray analy-
sis), more information may be obtained on the
structure alteration as well as subtle interactions
between functional groups.
2. Up to now, there have been no reports on PICT or other
parameter changes in microbial communities influenced
by antibiotics belonging to the bactericidal group, such as
β-lactam antibiotics. Indeed, the most often studied
bacteriostatic antibiotics are widely used in agriculture
and veterinary medicine. However, in human medicine, ß-
lactams are the most frequently used antibiotics, which
account for about 50–70% of total antibiotic in use
(Kümmerer 2009a). The peak concentrations of β-
lactams has been reported at lower micrograms per liter
level in hospital effluent (Kümmerer 2001; Watkinson et
al. 2009) and wastewater treatment plant (WWTP)
effluent (Watkinson et al. 2009), and at higher nano-
grams per liter level in environmental waters (Watkinson
et al. 2009). Though β-lactams are rapidly degraded
through hydrolysis and decarboxylation in laboratory
conditions (Längin et al. 2009), their concentrations in
WWTP effluent are still high compared with other
classes of antibiotics due to the much higher influent
concentrations (Watkinson et al. 2009). Therefore, the ß-
lactam class should not be ignored, especially in their
effects on resistance expansion at low concentrations in
the environment.
938
3. The existing results give contradictory conclusions on the
casual relationship between antibiotic input and resistance
development in soil, possibly due to differences in
indigenous microbial community structure, soil conditions
and experimental procedures. The presence of antibiotics is
perhaps the one out of many factors, e.g., the introduction
of resistance genes and selection pressure from other
pollutants such as heavy metals (Kong et al. 2006). Mask
effect brought by large quantities of resistance in the
manure could also account for the absence of detectable
antibiotic effects (Heuer et al. 2008).
4. Studies on antibiotic effects on soil ecological functions
are limited. Soil services other than nitrogen transfor-
mation have not been covered much. Current findings
seem to show the buffering capability of ecosystems
and the recovery from the inhibition of certain groups.
5. Rhizosphere is found to be an important place for
resistance development, but further studies are needed
to tackle the biological complexity within the rhizo-
sphere and pinpoint the mechanism behind this stimu-
lation phenomenon.
6. Antibiotics effects in aquatic environment are less studied
than in soil environment. However, the study is of
significance because of the wide coverage of water body
and the potential impacts of lower level antibiotics.
Similar to the soil ecosystem, resilience of the aquatic
ecosystem is implied in lab-scale SBR reactors when
studying their nitrogen/phosphorus removal abilities.
7. Antibiotic effects on ecosystems are related to its
concentration, bioavailability, exposure time and the
addition of substrates. Effects of multiple antibiotics
and the coexistence of antibiotics plus heavy metals
(Tuckfield and McArthur 2008; Stepanauskas et al.
2006), though often present in the natural habitat, have
not received enough notice so far. Their effects are
expected to be different from simple stacking of
individual antibiotic effects, according to the lab-scale
tests on pure isolates.
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