Post on 05-Feb-2023
FEMS Immunology and Medical Microbiology 41 (2004) 35–41
www.fems-microbiology.org
Persistence of a Staphylococcus aureus small-colony variantunder antibiotic pressure in vivo
Eric Brouillette a, Alejandro Martinez b, Bobbi J. Boyll b,Norris E. Allen b, Franc�ois Malouin a,*
a Centre d’�Etude et de Valorisation de la Diversit�e Microbienne (CEVDM), D�epartement de biologie,
Universit�e de Sherbrooke, Sherbrooke, Que., Canada J1K 2R1b Elanco Animal Health, Eli Lilly and Co., Greenfield, IN, USA
Received 30 September 2003; received in revised form 8 December 2003; accepted 28 December 2003
First published online 5 February 2004
Abstract
Staphylococcus aureus small-colony variants (SCVs) have been implicated in chronic and persistent infections. Bovine mastitis
induced by S. aureus is an example of an infection difficult to eradicate by conventional antimicrobial therapies. In this study, the
ability to colonize mouse mammary glands and persist under antibiotic treatment was assessed for S. aureus Newbould and an
isogenic hemB mutant, which exhibited the classical SCV phenotype. The hemB mutant showed a markedly reduced capacity to
colonize tissues. However, although the hemB mutant was as susceptible as S. aureus Newbould to cephapirin in vitro, it was over a
100 times more persistent than the parental strain in the mammary glands when 1 or 2 mg kg�1 doses were administrated. These
results suggest that, although the hemB mutant has a reduced ability to colonize mammary glands, the SCV phenotype may account
for the persistence of S. aureus under antibiotic pressure in vivo.
� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Staphylococcus aureus; hemB; Small-colony variant; SCV; Mastitis; Antibiotic resistance
1. Introduction
A link has been proposed between the presence ofsmall-colony variants (SCVs) of Staphylococcus aureus
and persistent and recurring infections, especially in
cases of human osteomyelitis and pulmonary infections
in cystic fibrosis [1]. S. aureus SCVs typically exhibit
slow-growing pinpoint colonies on agar, which are at-
tributable to a defective electron transport chain that
can be restored by either hemin or menadione supple-
mentation (see [2] for a review).Bovine mastitis is usually caused by microbial in-
tramammary infection (IMI). Multiple bacterial spe-
* Corresponding author. Tel.: +1-819-821-8000-1202; fax: +1-819-
821-8049.
E-mail address: francois.malouin@usherbrooke.ca (F. Malouin).
0928-8244/$22.00 � 2004 Federation of European Microbiological Societies
doi:10.1016/j.femsim.2003.12.007
cies can colonize the bovine mammary gland and
satisfactory cure rates are achieved by antimicrobial
treatment for the majority of these pathogens. How-ever, S. aureus IMIs are difficult to treat and relapsing
infections frequently occur following apparently suc-
cessful antimicrobial therapies [3]. S. aureus SCVs
have been isolated from bovine intramammary infec-
tions [4]. Nevertheless, to our knowledge, no study has
addressed so far the potential role of these phenotypic
variants in the persistence of infectious mastitis. The
present study reports the virulence of a geneticallydefined S. aureus SCV mutant in a model of mastitis
in the mouse and its persistence during antibiotic
therapy. We have found that, although the hemB
mutant showed a markedly reduced capacity to colo-
nize the mammary gland tissue, antibiotic treatment in
vivo proportionally kills more bacteria of the wild-
type than of the S. aureus SCVs.
. Published by Elsevier B.V. All rights reserved.
36 E. Brouillette et al. / FEMS Immunology and Medical Microbiology 41 (2004) 35–41
2. Materials and methods
2.1. Bacterial strains
Staphylococcus aureus strain Newbould 305 (ATCC29740), isolated from bovine mastitis [5], was used as the
parental strain in this study. The construction of S.
aureus hemB, a hemB insertion mutant derived from S.
aureus Newbould 305, is described at Section 3.1 and
was used as a representative of S. aureus SCV isolates.
In this report, the parental strain and its isogenic mutant
are denoted as S. aureus Newbould and S. aureus hemB,
respectively.
2.2. Growth of bacteria
Staphylococcus aureus was routinely grown using
Trypticase soy agar (TSA) and Trypticase soy broth
(TSB); TSA supplemented with 5% sheep red blood cells
was used to detect hemolytic activity. Mueller–Hinton II
cation-adjusted broth (MHBCA) was used to assessantibiotic activity. A semi-defined medium [8], contain-
ing either 10 mM glucose or 50 mM mannitol, was used
to determine utilization of these sugars as the sole car-
bon sources.
2.3. Hemolysin assay with growth medium
Bacteria were grown in TSB with or without heminsupplementation (1 lg ml�1). Following overnight
growth, A650 nm was measured and cells were removed by
centrifugation. Aliquots of supernatants were incubated
with sheep red blood cells for 1 h at 37 �C to allow lysis.
Red blood cell debris was pelleted by centrifugation and
released hemoglobin was measured at A540 nm. Hemolytic
activity was estimated based on the ratio of
A540 nm=A650 nm and was subsequently reported in termsof percentage of activity between strains or between
hemin-supplemented and non-supplemented cultures.
2.4. In vitro antibacterial activity
The minimal inhibitory concentrations (MICs) of
antibiotics were determined by a broth microdilution
technique, following the recommendations of theNCCLS [9]. The inoculum was 105–106 cfu (colony-
forming units) ml�1 and plates were incubated at 35 �Cfor 24 and 48 h for S. aureus Newbould and S. aureus
hemB, respectively, in order to allow maximal growth
for the controls without antibiotics. For measuring the
bactericidal action of cephapirin, bacteria were added
to MHBCA in culture tubes in the presence or absence
of cephapirin (4· MIC) and allowed to grow at 35 �C(225 rpm). Samples were taken at different time-points
for the determination of the cfu. The detection limit
was 100 cfu ml�1.
2.5. Bacterial invasion and persistence in cultured
mammalian cells
Normal mouse mammary gland epithelial cells
(NMuMG; ATCC CRL-1636) were grown as mono-layers by plating 5 · 105 cells in collagen-coated 24-well
plates (Becton Dickenson) in Dulbecco�s modified Ea-
gle�s medium (DMEM; Gibco-BRL), containing 10%
inactivated fetal bovine serum and 1% insulin. The
monolayers were grown to confluence overnight under
10% CO2 at 37 �C. After that, they were washed with
DMEM salt solution (Gibco-BRL) and incubated with
1 · 106 DMEM-washed bacteria grown overnight onTSA. Invasiveness was determined by exposing the
monolayers to bacteria for 3 h. Then, they were washed
and incubated an additional 30 min in a salt solution
containing 10 lg ml�1 lysostaphin [10]. Following ex-
tensive washing, the monolayers were lysed with 0.1%
Triton X-100 in distilled water. The lysate was serially
diluted and 50-ll aliquots were plated on TSA for de-
termining the cfu. Persistence in NMuMG cells wasdetermined using the same procedure, except that the
monolayers were incubated in complete media contain-
ing 10 lg ml�1 lysostaphin and assayed for intracellular
bacteria at 24, 48, 72 and 96 h time points.
2.6. Infectivity of S. aureus in the mouse mastitis model
The mouse mastitis model of infection used here isbased on that previously described by Chandler [11] and
adapted for infection with S. aureus Newbould (E.
Brouillette, B.G. Talbot, G. Grondin, F. Malouin,
Abstr. 102nd Gen. Meet. Am. Soc. Microbiol. 2002,
abstr. Z-56, 2002). Briefly, 1 h following removal of 12–
14 day-old offspring, lactating CD-1 mice were anes-
thetized with ketamine and xylazine at 87 and 13 mg
kg�1 body weight, respectively, and mammary glandswere inoculated under a binocular. Mammary ducts
were exposed by a small cut at the near ends of teats and
a 100-ll bacterial suspension, containing approximately
101 or 102 cfu in endotoxin-free phosphate-buffered sa-
line (PBS, Sigma), was injected through the teat canal
using a 33-gauge blunt needle. Two glands (fourth on
the right [R4] and fourth on the left [L4]) were inocu-
lated for each animal. Mammary glands were asepticallyharvested at the indicated times and the bacterial con-
tent was evaluated after tissue homogenization in 2 ml
of PBS, preparing serial logarithmic dilutions and plat-
ing on agar for cfu determination (detection limit of
approximately 50 cfu g�1 of gland).
2.7. Cephapirin treatment in the mouse mastitis model
Inoculation of mouse mammary glands was carried
out as described above and the infection allowed to
proceed for 24 h. Cephapirin in PBS was delivered into
E. Brouillette et al. / FEMS Immunology and Medical Microbiology 41 (2004) 35–41 37
the tail vein (200 ll) at t ¼ 0 and 10 h post-infection at 1,
2 or 6 mg kg�1 of weight. Each dose of cephapirin was
tested in separate experiments with non-treated controls.
2.8. Statistical analyses
Raw bacterial cfu counts were transformed in based-
10 logarithm values and used for the statistical analysis
of bacterial colonization between animal groups. Sta-
tistical significance was evaluated by the non-parametric
Kruskal–Wallis ANOVA test, with a post-hoc test for
multiple pairwise comparisons (Dwass–Steel) using the
StatsDirect software (version 2.2.7). P < 0:05 was con-sidered as statistically significant and non-significant
differences were denoted ‘‘N.S.’’.
3. Results
3.1. Insertional inactivation of the hemB gene
The gene hemB [6] from S. aureus Newbould was
cloned into a temperature-sensitive plasmid and dis-
rupted by homologous recombination. Specifically, the
PCR-amplified hemB gene was cloned into pBT2 [7] in
Escherichia coli. Plasmid pBT2 is a chloramphenicol-
resistant (Cmr), temperature-sensitive shuttle vector that
replicates in E. coli and S. aureus. Insertional inactiva-
tion was accomplished by cloning an ermA cassette intoa Bcl1 site in hemB in the recombinant plasmid. The
resulting plasmid was transferred for propagation into
S. aureus SA113 (res�). Plasmid DNA was then isolated
and used to transform S. aureus Newbould by electro-
poration. Homologous recombinants with the inser-
tional inactivated hemB gene were selected as Emr Cms
colonies that grew at 40 �C.
3.2. In vitro characterization of the hemB mutant and
validation of the SCV phenotype
(i) Respiratory deficiency and growth density. hemB
is the determinant for d-aminolevulinate dehydrase, an
essential enzyme in porphyrin biosynthesis converting
d-aminolevulnic acid to porphobilinogen [12]. Lacking
this enzyme, the hemB mutant does not synthesizeheme, resulting in a defective electron transport sys-
tem. The mutant is dependent on fermentative me-
tabolism for growth. S. aureus Newbould was able to
grow in a semi-defined medium containing either
glucose or mannitol as sole carbon source, growth on
the latter indicating a functioning electron transport
system to support respiration. In contrast, S. aureus
hemB could grow with glucose but not with mannitolas the sole carbon source, indicating respiratory defi-
ciency. Addition of 1 lg ml�1 of hemin to semi-
defined medium did support the growth of the hemB
mutant on mannitol (data not shown). In broth,
growth of the S. aureus hemB mutant reached a pla-
teau at a lower bacterial density compared to S. au-
reus Newbould (data not shown). The addition of
hemin (P 0.2 lg ml�1) restored the growth rate andthe capacity S. aureus hemB to reach a maximal
bacterial density equivalent to that of the parental
strain. The doubling times were calculated to be
22.6 ± 3.3 min for the wildtype strain and 53.3 ± 4.8
min for the hemB mutant in MHBCA.
(ii) Colonial morphology and hemolysin production.
After 48 h of incubation at 37 �C on TSA, colonies of S.
aureus Newbould hemB were approximately 1 mm indiameter, whereas colonies of the parent strain were 4
mm or larger in diameter. Colony size of the hemB
mutant was partially restored by the addition of hemin
(5 lg ml�1). S. aureus Newbould hemB was non-hemo-
lytic when streaked onto blood agar plates and incu-
bated overnight in 5% CO2 at 37 �C. The parental strainwas strongly hemolytic in this test. When examined for
extracellular hemolysin activity, the hemB mutantshowed only 3% of the activity of the parental strain
(data not shown). When the growth medium in this
experiment was supplemented with hemin (1 lg ml�1),
the hemolysin activity of the mutant strongly increased
(92%) as compared to the increase observed for the
parental strain (27%).
3.3. Bacterial invasion and persistence in mouse mam-
mary epithelial cells
Staphylococcus aureus Newbould and the hemB mu-
tant were compared for invasiveness in NMuMG epi-
thelial monolayer cell cultures. Based on recovery of cfu,
both strains were invasive on NMuMG cells, though the
invasiveness of the mutant was nearly fourfold higher
than that of the parent (Fig. 1(a)). Growth of S. aureuslikely occurred during the 3-h incubation period, as
evidenced by the fact that recovery of the hemB mutant
exceeded 100%. Compared to S. aureus Newbould, S.
aureus hemB also showed higher intracellular persistence
in NMuMG cells over time (Fig. 1(b)). Intracellular
survival after 96 h of incubation (cfut¼96 h (cfut¼0 h)�1)
was 0.16 for the hemB mutant and 0.032 for S. aureus
Newbould.
3.4. Infectivity in a mouse mastitis model
Overall, the mouse model of mastitis revealed similar
infection profiles for both S. aureus Newbould and
hemB strains (Fig. 2). For all inocula used, the expo-
nential phase of the infection took place mainly within
the first 12 h of infection, while maximal infection wasreached at 24 h in each case. In spite of this, an ap-
proximate three-log difference, in terms of bacterial
burden (cfu g�1 of gland) at 12 h and thereafter, was
Fig. 1. (a) Bacterial invasion of mouse mammary epithelial cells. NMuMG cells grown to confluence as monolayers were exposed to S. aureus
Newbould and S. aureus hemB for 3 h at 37 �C, washed, and then incubated with lysostaphin to remove extracellular bacteria. Intracellular bacteria
were enumerated following lysis as described in Section 2. Cfu recovered are reported as percentage of the original cfu added to the cell cultures. (b)
Intracellular persistence of bacteria. NMuMG cells were infected as above, except that infected cells were incubated for various periods (0–96 h) in
the presence of lysostaphin. Intracellular bacteria were enumerated following lysis as described in Section 2.
Fig. 2. Bacterial proliferation in the mouse mastitis model of infection.
Mice were inoculated intramammarly with approximately 102 cfu per
gland of S. aureus strain Newbould (d) or hemB (j). In a similar way,
an inoculum of 101 cfu per gland was also used for both strains and
medians are represented by the corresponding empty forms. For each
condition of infection, 4–10 mammary glands from a minimum of
three mice were harvested, homogenized and the bacterial cfu counts
were evaluated as described in Section 2.
38 E. Brouillette et al. / FEMS Immunology and Medical Microbiology 41 (2004) 35–41
found between the strains in favor of S. aureus New-
bould, indicating a markedly reduced capacity of strain
hemB to multiply in the mammary gland.
Table 1
MICs (in lg ml�1) for S. aureus Newbould and S. aureus hemB to different
Antibiotic Minimal inhibitory conce
S. aureus Newbould
24 h
Cephapirin 0.12
Ciprofloxacin 0.12
Erythromycin 0.25
Gentamicin 0.25–0.5
Gentamicin (5 lg ml�1 hemin) 0.25–0.5
Lysostaphin 2
Oxacillin 0.12
Rifampicin 0.008–0.015
Vancomycin 0.5
3.5. Antibiotic susceptibility
Similar MIC values were obtained for S. aureus
Newbould (24 h) and S. aureus hemB (48 h) with
cephapirin, ciprofloxacin, lysostaphin, oxacillin, rifam-
picin and vancomycin (Table 1). The exceptions were
erythromycin and gentamicin. An erythromycin resis-
tance gene (ermA) was used as a marker to construct the
S. aureus hemB mutant, and consequently, the bacte-rium exhibited a markedly higher MIC for this antibi-
otic. Besides, the defective electron transport chain of
the SCV affects the electrochemical gradient across the
bacterial membrane and thus reduces the penetration of
aminoglycoside antibiotics into cells [2]. Typical of the
SCV and respiratory deficient mutants, the gentamicin
MIC for hemB was 8–16 times higher than for the pa-
rental strain; addition of 5 lg ml�1 of exogenous heminrestored the susceptibility of S. aureus hemB to the level
of S. aureus Newbould. For the antibiotic susceptibility
tests, it should be noted that the S. aureus hemB mutant
needed to be incubated for 48 h to allow maximal
growth instead of 24 h for the parent strain, and this
because of its slower growth rate. For similar experi-
antibiotics as determined by a broth micro-dilution method
ntration (MIC)
S. aureus hemB
24 h 48 h
0.06 0.06–0.12
0.06 0.12–0.25
1–2 >16
2–4 4
0.12–0.25 0.25
1 1
0.06 0.06
0.008–0.015 0.015
0.5 0.5
E. Brouillette et al. / FEMS Immunology and Medical Microbiology 41 (2004) 35–41 39
ments, 72 h of incubation for hemB mutants have been
used by others [13]. However, similar MIC data were
obtained in the present study when using 72 h of incu-
bation instead of 48 h. Before conducting in vivo ex-
periments with cephapirin, the cidal activity of theantibiotic was also assessed against both S. aureus
strains. Cephapirin time-kill curves showed no difference
in the susceptibility of S. aureus Newbould and hemB as
determined by the reduction of cfu in time (Fig. 3).
Fig. 3. Susceptibility of bacteria to the bactericidal effect of cephapirin.
S. aureus strain Newbould (s) or hemB (�) were grown in MHBCA in
the presence of cephapirin (4· MIC; 0.5 lg ml�1) and the content of
cfu ml�1 was determined over time. The growth of bacteria without
antibiotic is represented on the figure by the corresponding solid
forms.
Fig. 4. Treatment of mouse mammary gland infection with cephapirin. M
Newbould or hemB (1.4–1.8· 102 cfu per gland) followed by cephapirin i.v. a
infected controls were carried out for each dose of cephapirin tested and th
between treatments are indicated.
3.6. Treatment of S. aureus mastitis with cephapirin
Both strains responded in a dose-dependent manner
to antibiotic treatment of mouse IMI with cephapirin.
However, the observed antibacterial effect was highlyreduced for S. aureus hemB compared to that measured
for Newbould (Fig. 4). S. aureus hemB was indeed rel-
atively more persistent upon cephapirin administration,
as evaluated after 24 h of infection (in terms of relative
reduction in cfu compared to untreated control). Spe-
cifically, the median log10 of cfu g�1 of gland was re-
duced by 3.17 (P < 0:001) and 0.88 (P ¼N.S.) for S.
aureus Newbould and hemB strains, respectively, when 1mg kg�1 doses of cephapirin were administrated. In the
same manner, the persistence of hemB to the antibac-
terial activity of cephapirin was also noted with 2 mg
kg�1 doses. When the cephapirin doses used were in-
creased to 6 mg kg�1, mammary glands of mice infected
with either S. aureus Newbould or S. aureus hemB were
found uninfected.
4. Discussion
Staphylococcus aureus mammary gland infections in
cows do not always readily respond to antibiotic treat-
ment and sometimes become persistent and recurring
ammary glands of lactating mice were infected with either S. aureus
dministration of 1, 2 and 6 mg kg�1 of weight at t ¼ 0 and 10 h. Non-
e data were pooled. Median values (bars) and statistical significance
40 E. Brouillette et al. / FEMS Immunology and Medical Microbiology 41 (2004) 35–41
[3,14]. The S. aureus SCV phenotype has been linked to
clinical cases of recurring infections in humans [15]. S.
aureus SCVs have also been isolated from cases of bo-
vine mastitis [4]. However, no study has yet demon-
strated a direct link between the SCV phenotype andpersistent cases of intramammary infections.
The aim of this study was to assess the possible role
of the S. aureus SCV phenotype in chronic IMIs that
occur in cows despite antimicrobial therapy. A mastitis
isolate, S. aureus Newbould 305, which has been used
previously for experimental IMIs of bovines [5,16–19],
was employed to confer a relevant genetic background
to construct the SCV hemBmutant. The mouse model ofinfectious mastitis used in this study was previously
characterized and validated in our laboratory for the
study of S. aureus IMI pathogenesis [20].
The S. aureus hemB mutant constructed here dis-
played classical S. aureus SCV properties. Compared to
the normal phenotype, the mutant produced smaller
colonies, was less hemolytic and less susceptible to
gentamicin, three properties that were reverted by sup-plemental hemin. The mutant�s resistance to gentamicin,
an aminoglycoside antibiotic, indicates a respiratory
deficiency typical of clinical SCVs [24–26]. Also, the
hemB mutant was more invasive and persistent than the
parental strain after exposure to mouse mammary epi-
thelial cells cultured in vitro. The increased intracellular
persistence is probably a consequence of the lowered
amount of a-toxin (hemolysin) produced [21,22] incombination with higher invasiveness, the result of an
increased production of fibronectin-binding proteins
[10,23].
Consistent with the slow-growing, non-hemolytic
phenotype of SCVs, the S. aureus hemB mutant had a
reduced ability to colonize mammary glands in the
mouse mastitis model of infection. This was regardless
of the fact that more hemB mutant bacteria were re-covered in time during in vitro experiments evaluating
the persistence of the pathogen within cultured mam-
mary epithelial cells. This can be explained by the fact
that the proportion of intracellular or extracellular
bacteria does not influence the total number of bacteria
recovered from the mammary tissue. Other authors [27]
have also observed a marked reduction in the capacity
of another S. aureus hemB mutant (constructed fromstrain Newman) to colonize kidneys 2 week after i.v.
administration. Paradoxically, in the mouse model of
mastitis, we demonstrated a reduced ability for cepha-
pirin to control a bacterial infection caused by the S.
aureus hemB mutant as compared to a similar infection
caused by the fully virulent strain Newbould. This was
also despite the fact that in vitro susceptibilities of both
strains to the bactericidal action of cephapirin werenearly identical. In contrast to our results, in an endo-
carditis model, others found that a S. aureus 8325-4
hemB mutant was as susceptible as its parental strain to
elimination by oxacillin treatment [28]. It was proposed
that target organs may have been repleted with hemin
during the course of endocarditis and thus restored the
wild-type phenotype. Results from the latter study in-
dicate that the consequences of a hemB mutation on S.
aureus may vary between types of infection.
The results obtained in the mouse model suggest that
the SCV phenotype may be favored in vivo when the
antimicrobial concentration is high enough to propor-
tionally kill more bacteria harboring the normal phe-
notype. The slower growth rate of S. aureus hemB did
not affect its susceptibility to cephapirin in vitro and
cannot solely explain the persistence of the hemBmutantin vivo. For most antibiotics, intracellular killing of
bacteria is much less efficient than extracellular killing,
and SCVs appear to be able to invade and survive within
non-phagocytic cells more successfully than the normal
phenotype. It remains to be determined whether the
persistence of the hemB mutant observed here in the
mastitis model of infection is due to intracellular SCVs.
Staphylococcus aureus, having either a normal orSCV phenotype, is able to invade non-phagocytic cells
in vitro. However, in the case of SCVs, a reduced
hemolysin production may extend the capacity of bac-
teria to remain within eucaryotic cells, avoiding the
immune system and antibiotics for a longer period of
time. The instability of naturally occurring S. aureus
SCVs, which frequently revert to the normal phenotype
in vitro [29], suggests that the pathogen may becomequiescent for a time before re-expressing a virulent
phenotype to provoke a resurgence of infection. In the
present study, the results collected from our mouse
model of S. aureus mastitis support the notion that
hemin-auxotrophic SCVs are less susceptible to antibi-
otic therapy, thus providing a partial explanation for
why S. aureus-induced bovine mastitis is so difficult to
eradicate.
Acknowledgements
The authors thank R. Br€uckner for providing pBT2.This study was partly supported by Grant No. MOP-
57701 to FM from the Canadian Institutes for Health
Research.
References
[1] von Eiff, C., Proctor, R.A. and Peters, G. (2000) Staphylococcus
aureus small colony variants: formation and clinical impact. Int. J.
Clin. Pract. (Supp), l44–149.
[2] Proctor, R.A., Kahl, B., von Eiff, C., Vaudaux, P.E., Lew, D.P.
and Peters, G. (1998) Staphylococcal small colony variants have
novel mechanisms for antibiotic resistance. Clin. Infect. Dis. 27
(Suppl. 1), S68–S74.
E. Brouillette et al. / FEMS Immunology and Medical Microbiology 41 (2004) 35–41 41
[3] Gruet, P., Maincent, P., Berthelot, X. and Kaltsatos, V. (2001)
Bovine mastitis and intramammary drug delivery: review and
perspectives. Adv. Drug Deliv. Rev. 50, 245–259.
[4] Sompolinsky, D., Cohen, M. and Ziv, G. (1974) Epidemiological
and biochemical studies on thiamine-less dwarf-colony variants of
Staphylococcus aureus as etiological agents of bovine mastitis.
Infect. Immun. 9, 217–228.
[5] Newbould, F.H. (1974) Antibiotic treatment of experimental
Staphylococcus aureus infections of the bovine mammary gland.
Can. J. Comp. Med. 38, 411–416.
[6] Kafala, B. and Sasarman, A. (1994) Cloning and sequence
analysis of the hemB gene of Staphylococcus aureus. Can. J.
Microbiol. 40, 651–657.
[7] Bruckner, R. (1997) Gene replacement in Staphylococcus carnosus
and Staphylococcus xylosus. FEMS Microbiol. Lett. 151, 1–8.
[8] Lascelles, J. (1979) Heme-deficient mutants of Staphylococcus
aureus. Methods Enzymol. 56, 172–178.
[9] National Committee for Clinical Laboratory Standards. Methods
for Dilution Antimicrobial Susceptibility Tests for Bacteria that
grow Aerobically, second edition. Approved Standard M7-A2.
NCCLS, Villanova, PA, 1997.
[10] Vaudaux, P., Francois, P., Bisognano, C., Kelley, W.L., Lew,
D.P., Schrenzel, J., Proctor, R.A., McNamara, P.J., Peters, G. and
von Eiff, C. (2002) Increased expression of clumping factor and
fibronectin-binding proteins by hemB mutants of Staphylococcus
aureus expressing small-colony variant phenotypes. Infect. Im-
mun. 70, 5428–5437.
[11] Chandler, R.L. (1970) Experimental bacterial mastitis in the
mouse. J. Med. Microbiol. 3, 273–282.
[12] Kafala, B. and Sasarman, A. (1997) Isolation of the Staphylococ-
cus aureus hemCDBL gene cluster coding for early steps in heme
biosynthesis. Gene 199, 231–239.
[13] Baumert, N., von Eiff, C., Schaaff, F., Peters, G., Proctor, R.A.
and Sahl, H.G. (2002) Physiology and antibiotic susceptibility of
Staphylococcus aureus small-colony variants. Microb. Drug Re-
sist. 8, 253–260.
[14] Sears, P.M. and McCarthy, K.K. (2003) Management and
treatment of staphylococcal mastitis. Vet. Clin. North Am. Food
Anim Pract. 19, 171–185, vii.
[15] Proctor, R.A., Vesga, O., Otten, M.F., Koo, S.P., Yeamen, M.R.,
Sahl, H.G. and Bayer, A.S. (1996) Staphylococcus aureus small-
colony variants cause persistent and resistant infections. Chemo-
therapy 42, 47–52.
[16] Balaban, N., Collins, L.V., Cullor, J.S., Hume, E.B., Medina-
Acosta, E., Vieira, d.M., O�Callaghan, R., Rossitto, P.V., Shirtliff,
M.E., Serafim, d.S., Tarkowski, A. and Torres, J.V. (2000)
Prevention of diseases caused by Staphylococcus aureus using the
peptide RIP. Peptides 21, 1301–1311.
[17] Gudding, R., McDonald, J.S. and Cheville, N.F. (1984) Patho-
genesis of Staphylococcus aureus mastitis: bacteriologic, histologic
and ultrastructural pathologic findings. Am. J. Vet. Res. 45, 2525–
2531.
[18] Hensen, S.M., Pavicic, M.J., Lohuis, J.A., de Hoog, J.A. and
Poutrel, B. (2000) Location of Staphylococcus aureus within the
experimentally infected bovine udder and the expression of
capsular polysaccharide type 5 in situ. J. Dairy Sci. 83, 1966–
1975.
[19] Schukken, Y.H., Leslie, K.E., Barnum, D.A., Mallard, B.A.,
Lumsden, J.H., Dick, P.C., Vessie, G.H. and Kehrli, M.E. (1999)
Experimental Staphylococcus aureus intramammary challenge in
late lactation dairy cows: quarter and cow effects determining the
probability of infection. J. Dairy Sci. 82, 2393–2401.
[20] Brouillette, E., Talbot, B.G. and Malouin, F. (2003) The
fibronectin-binding proteins of Staphylococcus aureus may pro-
mote mammary gland colonization in a lactating mouse model of
mastitis. Infect. Immun. 71, 2292–2295.
[21] Balwit, J.M., van Langevelde, P., Vann, J.M. and Proctor, R.A.
(1994) Gentamicin-resistant menadione and hemin auxotrophic
Staphylococcus aureus persist within cultured endothelial cells. J.
Infect. Dis. 170, 1033–1037.
[22] Vann, J.M. and Proctor, R.A. (1988) Cytotoxic effects of ingested
Staphylococcus aureus on bovine endothelial cells: role of S. aureus
alpha-hemolysin. Microb. Pathog. 4, 443–453.
[23] Dziewanowska, K., Patti, J.M., Deobald, C.F., Bayles, K.W.,
Trumble, W.R. and Bohach, G.A. (1999) Fibronectin binding
protein and host cell tyrosine kinase are required for internaliza-
tion of Staphylococcus aureus by epithelial cells. Infect. Immun.
67, 4673–4678.
[24] Abele-Horn, M., Schupfner, B., Emmerling, P., Waldner, H. and
Goring, H. (2000) Persistent wound infection after herniotomy
associated with small-colony variants of Staphylococcus aureus.
Infection 28, 53–54.
[25] Kahl, B., Herrmann, M., Everding, A.S., Koch, H.G., Becker, K.,
Harms, E., Proctor, R.A. and Peters, G. (1998) Persistent infection
with small-colony variant strains of Staphylococcus aureus in
patients with cystic fibrosis. J. Infect. Dis. 177, 1023–1029.
[26] von Eiff, C., Becker, K., Metze, D., Lubritz, G., Hockmann, J.,
Schwarz, T. and Peters, G. (2001) Intracellular persistence of
Staphylococcus aureus small-colony variants within keratinocytes:
a cause for antibiotic treatment failure in a patient with darier�sdisease. Clin. Infect. Dis. 32, 1643–1647.
[27] Jonsson, I.M., von Eiff, C., Proctor, R.A., Peters, G., Ryden,
C. and Tarkowski, A. (2003) Virulence of a hemB mutant
displaying the phenotype of a Staphylococcus aureus small-
colony variant in a murine model of septic arthritis. Microb.
Pathog. 34, 73–79.
[28] Bates, D.M., Eiff, C.C., McNamara, P.J., Peters, G., Yeaman,
M.R., Bayer, A.S. and Proctor, R.A. (2003) Staphylococcus aureus
menD and hemB mutants are as infective as the parent strains, but
the menadione biosynthetic mutant persists within the kidney.
J. Infect. Dis. 187, 1654–1661.
[29] Massey, R.C., Buckling, A. and Peacock, S.J. (2001) Phenotypic
switching of antibiotic resistance circumvents permanent costs in
Staphylococcus aureus. Curr. Biol. 11, 1810–1814.