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.

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