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Applied and Environmental Microbiology, June 2004, p. 3754-3757, Vol. 70, No. 6
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.6.3754-3757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Diverse Tetracycline Resistance Genotypes of Megasphaera elsdenii Strains Selectively Cultured from Swine Feces
Thaddeus B. Stanton,1* Jennifer S. McDowall,2 and Mark A. Rasmussen1
Pre-Harvest
Food Safety and Enteric Diseases Research Unit, National Animal Disease
Center, Agricultural Research Service, U.S. Department of
Agriculture, Ames, Iowa
50010-0070,1
Division of Molecular
Genetics, University of Glasgow, Glasgow G12 8QQ, United
Kingdom2
Received 22 December 2003/
Accepted 26 February 2004
 |
ABSTRACT
|
|---|
A
total of 30 Megasphaera elsdenii strains, selectively isolated
from the feces of organically raised swine by using Me109 M medium, and
one bovine strain were analyzed for tetracycline resistance genotypic
and phenotypic traits. Tetracycline-resistant strains carried
tet(O), tet(W), or a tet gene mosaic of
tet(O) and tet(W). M. elsdenii strains
carrying tet(OWO) genes exhibited the highest tetracycline
MICs (128 to >256 µg/ml), suggesting that
tet(O)-tet(W) mosaic genes provide the selective
advantage of greater tetracycline resistance for this species. Seven
tet genotypes are now known for M. elsdenii, an
archetype commensal anaerobe and model for tet gene evolution
in the mammalian intestinal
tract.
|
INTRODUCTION
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|---|
Megasphaera elsdenii is a commensal (mutualist) species in the
gastrointestinal tracts of ruminant and nonruminant mammals, including
humans (7,
25,
26). This anaerobic
bacterium contributes to the overall metabolism that takes place in
those microbial ecosystems
(2,
5,
13,
15). M. elsdenii
has been the focus of both prebiotic and probiotic applications for
improving animal health
(10,
12,
19,
27).
In a recent
study of intestinal bacteria resistant to tetracycline, we detected
resistant M. elsdenii strains at high population levels
(approximately 107 CFU/g) in cecal samples from healthy
swine (24). Eight strains
were isolated and characterized. The M. elsdenii strains are
highly resistant to chlortetracycline (MIC = 256 to
>256 µg/ml) and carry one of two
"tet(OWO)" genes for tetracycline resistance.
[Throughout the manuscript, "tet(OW)" and
"tet(OWO)" are used as convenient, practical
terms for describing M. elsdenii recombinant tet
genes and genotypes. As noted previously
(24), these
designations are not recognized under present tet
classification guidelines
(14). It is our hope that
future guidelines will be developed to accommodate these novel
interclass hybrid genes.] These tet genes are interclass
mosaic genes apparently formed by double-crossover recombinations
between tet(O) and tet(W) genes.
Our previous
study used a nutritionally complex medium with high concentrations of
chlortetracycline and, thus, was biased to select
tetracycline-resistant M. elsdenii strains. In this study,
Me109M medium was developed and used to select M. elsdenii
strains without using chlortetracycline. The goals were twofold: first,
to obtain tetracycline-sensitive strains of M. elsdenii useful
both for investigating tet gene transfer and for probiotic
applications, and second, to discover whether or not M.
elsdenii strains have additional tet
genotypes.
|
Selective isolation of M. elsdenii—Me109M medium.
|
|---|
On the basis of previous studies
(4-6,
9,
14,
16,
17,
19-21)
and preliminary experiments in our laboratory, Me109M medium was
developed to selectively culture M. elsdenii. Me109M contained
(per liter): tryptone-peptone, 4 g; yeast extract,
2 g; salts solution A (6 g of
K2HPO4/liter of water), 40 ml; salts solution B
[6 g of KH2PO4, 12 g of
(NH4)2SO4, 12 g of NaCl,
1.2 g of MgSO4-7H2O,
0.6 g of CaCl2/liter of water], 40 ml;
Na-DL-lactate syrup (60% [wt/wt]), 12.7 ml;
resazurin solution (0.1% [wt/vol]), 1 ml;
L-cysteine-HCl, 0.5 g; distilled water, 900 ml;
Difco Bacto agar, 12 g. The pH of the medium was adjusted to
5.0. A total of 10 ml of a monensin solution (5 mg/ml of ethanol) was
added to the autoclaved medium. Agar plates of medium were prepared
aerobically and, at least 2 days before inoculation, transferred into a
Coy anaerobic chamber at room temperature.
M. elsdenii
strains were isolated under anoxic conditions from fecal samples of 10
grower-phase (40- to 50-kg) swine purchased from two Iowa farms that
have raised animals organically (National Organic Foods Production Act;
Alternative Farming Systems Information Center
[www.nalusda.gov/afsic/ofp]),
without antibiotic feeding, for at least 4 years. The swine were housed
at the National Animal Disease Center, with no exposure to other
animals, in chemically decontaminated buildings with strict entry
requirements for human personnel. The animals were supplied water ad
libitum and fed their original organic diet, which was free of
detectable tetracycline
(3).
M.
elsdenii population densities averaged 2.4 x
108 CFU/g of feces (wet weight), and there were no
significant differences in counts between animals from the two farms.
M. elsdenii colonies appeared as large (2- to 3-mm-diameter),
yellow-white, dome-shaped colonies after 72 h of incubation
at 38°C and contained large cocci in pairs or chains. These
colonies represented approximately one-third of the colonies growing on
Me109M agar and 0.4% of the total cultivable fecal bacteria
(data not presented). Me109M is highly selective for M.
elsdenii and has been used to detect population levels
of this species as low as 104 CFU/g of feces (T.
B. Stanton, unpublished data). A total of 30 strains (3
from each animal) were cloned by subculturing and confirmed to be
M. elsdenii on the basis of physiological properties
and 16S rRNA-V3 sequence analysis
(24).
|
M. elsdenii tetracycline-resistant genotypes.
|
|---|
The 30 swine strains and bovine strain
Fern, previously isolated during unrelated studies, were analyzed by
PCR assays (24) to
differentiate known M. elsdenii tetracycline resistance genes
(Fig.
1; Table
1). Nine strains did not contain detectable tet genes. The
tet(OWO)-1 gene identified previously in swine M.
elsdenii strain 7-11 (Fig.
1) was not detected in any
of the isolates. A total of 11 strains had the
tet(OWO)-2 genotype reported previously for M.
elsdenii strain 14-14 (Fig.
1; Table
1). Another 11 strains
carried genes—tet(O), tet(W), and
tet(OW)—not previously detected for M.
elsdenii. The tet(OW) genes apparently originate from
single-crossover recombinations between tet(O) and
tet(W).
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|
FIG. 1. Schematic
depiction of PCR assays for differentiating M. elsdenii
tetracycline-resistant genotypes. Strain and tetracycline resistance
genotype designations are given at the left of the figure. Checkered
regions of genes have high-level sequence identity with
tet(O); open regions have high-level sequence identity with
tet(W). Products of PCR amplification are depicted by
double-headed arrows. Crossover regions of the genes were established
by sequence
analysis.
|
|
Sequence comparisons revealed differences
between the strain 27-51 and 25-51 tet(OW) genes;these have been designated tet(OW)-1 and
tet(OW)-2, respectively. In the strain 25-51
tet(OW)-2 gene, the
tet(O)-to-tet(W) crossover position occurs at base
position 243, resembling that of strain 14-14 (Fig.
1). The corresponding
crossover position of the strain 27-51 tet(OW)-1 gene
at base position 225 is similar to that of strain 7-11 (Fig.
1).
The
tet(W) genes of M. elsdenii strains 25-50 and 29-55
likely have different origins and have been designated, respectively,
tet(W)-1 and tet(W)-2. When the
Tet(W) portions (356-amino-acid segments) common to M.
elsdenii Tet(W), Tet(OW), and Tet(OWO) proteins are compared, the
amino acid sequences fall into two main clusters marked by a bootstrap
support value of 99 (Fig.
2). Sequences between the two clusters differ by 11 amino acids. One
cluster of segments is comprised of M. elsdenii Tet(W),
Tet(OW), and Tet(OWO) sequences. The sequences are identical,
suggesting that recombination between a tet gene identical to
tet(W)-2 of M. elsdenii strain 29-55 and a
tet(O) gene produced the presently known mosaic genes of
M. elsdenii. These sequences do not match any known Tet(W)
sequences of other intestinal species. By contrast, not only the M.
elsdenii 25-50 Tet(W)-1 segment (Fig.
2) but also the complete
25-50 Tet(W)-1 sequence is identical to those of Tet(W) proteins of
Butyrivibrio fibrisolvens and Mitsuokella
multiacidus, suggesting that these tet(W)
genes share a common origin and may be communicable among these
intestinal anaerobes. A larger sample of tet(W) sequences from
diverse bacterial species will undoubtedly increase the robustness of
the tree and could provide further insight into horizontal gene flow
among species in the intestinal tract.
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|
FIG. 2. Phylogenetic
analysis of a Tet(W) common region (365 amino acids) shared
by various species. GenBank accession numbers for nucleotide sequences
used to derive amino acid sequences are given in parentheses. The
unrooted neighbor-joining tree was generated using Mega2
(11). A Poisson
substitution model was applied to distance calculations. Branches are
labeled by bootstrap values from 10,000 replicates. Bar, number of
substitutions per amino
acid.
|
|
Seven tetracycline
resistance genotypes of M. elsdenii have now been detected in
these and previous studies
(24). M.
elsdenii appears to be a commensal "warehouse" for
tet(W), recombinant tet(O)-tet(W), and
perhaps tet(O) genes in the mammalian intestinal tract.
Whether M. elsdenii cells are the site for tet(O) and
tet(W) recombination or are the recipients of mosaic
tet genes remains unclear
(24).
|
Tetracycline MICs of M. elsdenii strains.
|
|---|
Tetracycline MICs for M.
elsdenii strains were determined by the agar dilution method
according to NCCLS recommendations
(18). All strains
carrying tet genes exhibited tetracycline antimicrobial MICs
that were 4- to >100-fold greater than those exhibited for
strains lacking the genes (Table
2). Of the strains, 11 carrying mosaic tet(OWO) genes consistently
exhibited the highest tetracycline MICs (128 to >256
µg/ml) (Table 2).
This finding implies that recombinant tet(O)-tet(W)
genes provide a selective advantage of increased tetracycline
resistance to M. elsdenii cells. An important test of this
hypothesis will be to examine the resistance properties of the various
tet genes in an isogenic background in, for example, the same
M. elsdenii strain.
Both tetracycline-sensitive and
tetracycline-resistant M. elsdenii strains were isolated from
the same fecal dilutions from every organically raised animal, an
indication that both types are present at population levels of
107 to 108 CFU/g of feces. Previously, M.
elsdenii strains exhibiting high tetracycline MIC levels and
carrying tet(OWO) genes were conservatively estimated at
107 CFU/g of cecal contents of conventionally raised swine
regardless of whether or not the animals were fed chlortetracycline
(24).
Tetracycline-resistant M. elsdenii strains persist at high
population levels in swine in the absence of antibiotic use.
Due
to the large diversity of uncultivated or difficult-to-culture
commensal anaerobes, it has been suggested that one or a few bacterial
species might serve as useful indicators in the analysis of reservoir
populations (7a).
Escherichia coli and Bacteroides and
Enterococcus spp. have been used to monitor the flow and
persistence of antibiotic resistance determinants within and between
intestinal ecosystems (1,
8,
22,
23,
28,
29). M. elsdenii
is a common anaerobe in the intestinal tracts of both ruminant and
nonruminant mammals, including humans. Currently known intestinal
Megasphaera comprise only one species, M. elsdenii.
M. elsdenii strains grow rapidly on simple anaerobic culture
medium, can be selectively isolated on Me109M agar, and have easily
recognizable cell and colony morphologies. M. elsdenii strains
are among the most numerous tetracycline-resistant populations in swine
intestinal tracts (24).
On the basis of these considerations, M. elsdenii offers
several attractive features as an indicator or archetype commensal
species for monitoring the antibiotic resistance status and reservoir
potential of the intestinal
microbiota.
|
Nucleotide sequence accession numbers.
|
|---|
The tet
gene sequences of five M. elsdenii strains were determined and
have been deposited in GenBank under the following accession numbers:
strain Fern, tet(O)
(AY485123);
strain 25-50, tet(W)-1
(AY485125);
strain 29-55, tet(W)-2
(AY485124);
strain 27-51, tet(OW)-1
(AY485126);
strain 25-51, tet(OW)-2
(AY485122).
|
ACKNOWLEDGMENTS
|
|---|
We
acknowledge Sam Humphrey for consistently competent technical support.
Brooke Peterson-Burch provided links to Mega2 software and insights
regarding computer analysis of Tet(W) phylogeny for which we are
grateful. We thank Tom Casey and Brooke Peterson-Burch for helpful,
in-depth reviews of the manuscript.
Mention of trade names or
commercial products in this article is solely for the purpose of
providing specific information and does not imply recommendation or
endorsement by the U.S. Department of
Agriculture.
|
FOOTNOTES
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* Corresponding
author. Mailing address: USDA Agriculture Research Service, National Animal Disease Center, 2300 N. Dayton Rd., P.O. Box 70, Ames, IA 50010-0070. Phone: (515) 663-7495. Fax: (515) 663-7458. E-mail: tstanton{at}nadc.ars.usda.gov. 
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Applied and Environmental Microbiology, June 2004, p. 3754-3757, Vol. 70, No. 6
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.6.3754-3757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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Abstract
Introduction
