International Journal of Antimicrobial Agents 32 (2008) 207–220

Review

A bioinformatic approach to understanding antibiotic resistance in

intracellular bacteria through whole genome analysis
Silpak Biswas, Didier Raoult, Jean-Marc Rolain ∗
URMITE UMR 6236, CNRS IRD, Faculté de Médecine et de Pharmacie, Université de la Méditerranée, 27 Bd Jean Moulin,
13385 Marseille Cedex 05, France
Received 19 March 2008; accepted 19 March 2008

Abstract
Intracellular bacteria survive within eukaryotic host cells and are difficult to kill with certain antibiotics. As a result, antibiotic resistance
in intracellular bacteria is becoming commonplace in healthcare institutions. Owing to the lack of methods available for transforming these
bacteria, we evaluated the mechanisms of resistance using molecular methods and in silico genome analysis. The objective of this review
was to understand the molecular mechanisms of antibiotic resistance through in silico comparisons of the genomes of obligate and facultative
intracellular bacteria. The available data on in vitro mutants reported for intracellular bacteria were also reviewed. These genomic data were
analysed to find natural mutations in known target genes involved in antibiotic resistance and to look for the presence or absence of differ-
ent resistance determinants. Our analysis revealed the presence of tetracycline resistance protein (Tet) in Bartonella quintana, Francisella
tularensis and Brucella ovis; moreover, most of the Francisella strains possessed the blaA gene, AmpG protein and metallo-␤-lactamase family
protein. The presence or absence of folP (dihydropteroate synthase) and folA (dihydrofolate reductase) genes in the genome could explain
natural resistance to co-trimoxazole. Finally, multiple genes encoding different efflux pumps were studied. This in silico approach was an
effective method for understanding the mechanisms of antibiotic resistance in intracellular bacteria. The whole genome sequence analysis will
help to predict several important phenotypic characteristics, in particular resistance to different antibiotics. In the future, stable mutants should
be obtained through transformation methods in order to demonstrate experimentally the determinants of resistance in intracellular bacteria.
© 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Keywords: Antibiotic resistance; Genomics; Sequence analysis; Intracellular bacteria; In silico; In vitro mutant

1. Introduction Treating bacterial infections is increasingly complicated

Intracellular bacteria are defined by their capacity to
survive and live inside eukaryotic host cells. As a result,
they have developed diverse strategies to survive within this
compartment. These bacteria are responsible for enormous
morbidity and mortality worldwide and are difficult to kill
with certain antibiotics. The barrier to antibiotic treatment of
obligate intracellular bacteria is the difference between the
localisation of antibiotics within the cellular compartments
of infected cells and the localisation of the bacteria [1].
∗ Corresponding author. Present address: Unité des Rickettsies, CNRS
UMR 6020, IFR 48, Faculté de Médecine et de Pharmacie, Université de la
Méditerranée, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France.
Tel.: +33 4 91 38 55 17; fax: +33 4 91 83 03 90.
E-mail address: jm.rolain@medecine.univ-mrs.fr (J.-M. Rolain).
by the ability of bacteria to develop resistance to different
antibiotics. Resistance to antibiotics can be caused by a
variety of mechanisms: (i) the presence of an enzyme that
inactivates the antimicrobial agent; (ii) a mutation in the target
of the antimicrobial agent that reduces its binding capacity;
(iii) post-transcriptional and post-translational modification
of the target of the antimicrobial agent, which reduces its
binding capacity; (iv) reduced uptake of the antimicrobial
agent; and (v) active efflux of the antimicrobial agent [2].
Bacteria can develop resistance to antibiotics through
two genetic processes: (i) mutation and selection (vertical
gene transfer); and (ii) exchange of genes between strains
and species (horizontal gene transfer). Among intracellular
bacteria, antibiotic resistance is primarily due to spontaneous
mutations or multiple mutations in the bacterial genome (i.e.
vertical gene transfer). To date, there is only one example of

0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2008.03.017
208 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220

horizontal gene transfer conferring resistance to tetracycline

among intracellular bacteria, namely Chlamydia suis [3].
In Mycobacterium tuberculosis, the causative agent of
tuberculosis, all of the drug resistance determinants are
chromosomally encoded, arising exclusively through the
acquisition and maintenance of spontaneous chromosomal
mutations in target or complementary genes [4]. Resistance-
associated point mutations, deletions or insertions in M.
tuberculosis have been previously described for all first-line
drugs (e.g. isoniazid, rifampicin, pyrazinamide, ethambutol
and streptomycin) in addition to several second-line and
newer drugs (e.g. ethionamide, fluoroquinolones, macrolides
and nitroimidazopyrans) [5].
The objective of this review was to present an overview
of the molecular evidence for bacterial resistance to
antimicrobial agents among intracellular bacteria using a
bioinformatic analysis of whole genome sequences and in
silico analysis of target genes.
2. Intracellular behaviour of bacteria and antibiotic
activity
The intracellular localisation of some bacteria remains
a critical point explaining the failure of some antibiotic
treatments in infected hosts. Parasites that multiply only
within eukaryotic cells are obligate intracellular pathogens,
whereas facultative intracellular pathogens can also multiply
in cell-free models [6].
Four categories of mechanisms exist to explain the sur-
vival of intracellular bacteria: (a) survival in the cytoplasm
Fig. 1. Lifestyles of intracellular bacteria: (1) bacterial escape into the
cytosol after exit from the endosomal compartment (e.g. Rickettsia, Shigella,
Listeria); (2) survival in non-fused phagosomes (e.g. Bartonella, Brucella,
Legionella); (3) segregation from the endolytic route and formation of a
unique inclusion vacuole (e.g. Chlamydia) and (4) survival by fusion with
the lysosome (e.g. Coxiella, Tropheryma, Francisella).
after exit from an endosomal compartment with or without
fusion of the phagosomal vacuole with lysosomes (e.g.
Rickettsia, Shigella and Listeria); (b) survival in non-fused
phagosomes (e.g. Bartonella, Brucella and Legionella); (c)
survival in fused phagosomes (e.g. Chlamydia); and (d) sur-
vival in fused phagolysosomes (e.g. Coxiella, Tropheryma
and Francisella) (Fig. 1).
Antibiotic activity against intracellular bacteria depends
on several factors, including pharmacodynamic and phar-
macokinetic properties of antibiotics. First, in order to
be active, antibiotics must reach the infected cells in
their tissue compartments via the systemic route. Second,
antibiotics need to reach and concentrate within intracellular
compartments. The intracellular to extracellular ratio (C/E)
is a very important factor and can be determined by several
methods, including radiometric, fluorometric and chemical

Table 1
Antibiotic susceptibility results for various intracellular bacteria
Intracellular bacteria Antibiotic

ERY AMG TET QUI CHL SXT RIF BL

Bartonella spp. S S S S S S S S
Tropheryma whipplei S S S R S S S S
Francisella tularensis S/R S S S S R S R
Rickettsia spp.
Typhus group
R. prowazekii S R S S S R S R
R. typhi S R S S S R S R
Spotted fever group
R. conorii subgroup
R. conorii, R. rickettsii R R S S S R S R
R. massiliae subgroup
R. massiliae, R. montanensis R R S S S R R R
Ehrlichia spp.
E. canis R R S R R R S R
E. chaffeensis R R S R R R S R
Wolbachia spp. R R S S/R R R S R
Coxiella burnetii S/R R S S R S S R
Brucella spp. S/R S S S R R S R
ERY, erythromycin; AMG, aminoglycosides; TET, tetracycline; QUI, quinolones; CHL, chloramphenicol; SXT, sulfamethoxazole/trimethoprim (co-
trimoxazole); RIF, rifampicin; BL, ␤-lactams; S, susceptible; R, resistant.
 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220
techniques. For example, the fluoroquinolones accumulate
within phagocytes with a C/E ratio of 6:7 for granulo-
cytes, 3:4 for macrophages and ca. 2:1 for epithelial cells
[7–9]. Third, antibiotics should remain active within the
targeted intracellular compartment, without inactivation by
cellular metabolism and/or deleterious effect of pH [6,10].
Some antibiotics are more effective at neutral or basic pH
values (e.g. fluoroquinolone compounds) but others (e.g.
rifampicin) are more effective at acidic pH values [6].
3. Overview of natural antibiotic susceptibility
among intracellular bacteria
The antibiotic susceptibility of facultative intracellular
bacteria can be assessed in cell-free systems using minimum
inhibitory concentration (MIC) determination methods;
for obligate intracellular bacteria, however, antibiotic
Fig. 2. Phylogenetic tree based on 16S sequences of intracellular bacteria used in this study.
209
susceptibility should be determined only in cell models.
The choice of cell system depends on each pathogen, but
in general cell lines that are easy to obtain and grow are
used (e.g. Vero, L929 or MRC5 cells) [6]. The methods for
evaluating antibiotic susceptibility vary with the nature of
the intracellular pathogen [11]. One important technique
is enumeration of viable intracellular microorganisms
(colony-forming unit or plaque assay) after various times of
antibiotic exposure compared with drug-free controls (e.g.
Rickettsia, Coxiella) [6]. Other methods of evaluation have
also been used, including determination of the percentage
of infected cells, flow cytometry, immunofluorescence tech-
niques, luciferase techniques and quantitative polymerase
chain reaction (PCR) [7,12]. There are very few reports of
antibiotic susceptibility testing among fastidious bacteria, as
the methods are time consuming and labour intensive.
Table 1 summarises the natural susceptibility to antibi-
otics among intracellular bacteria. Bacteria of the genus
210 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220
Bartonella are susceptible to all antibiotics in vitro, including
␤-lactams, aminoglycosides, chloramphenicol, tetracyclines,
macrolides, rifampicin, fluoroquinolones and co-trimoxazole
[13,14]. Tropheryma whipplei displays a homogeneous pat-
tern of antibiotic susceptibility in axenic medium [15], with
almost all antibiotics showing at least some activity, except
fluoroquinolones [16]. Francisella tularensis strains are
susceptible to streptomycin, gentamicin, doxycycline, chlo-
ramphenicol and quinolones but show heterogeneous sus-
ceptibility to erythromycin [17,18]. Rickettsia are naturally
resistant to ␤-lactams, aminoglycosides and co-trimoxazole.
The typhus group is susceptible to erythromycin, whereas the
spotted fever group is not [19,20] (Table 1).
In vitro antibiotic susceptibility studies have shown that
various species of Ehrlichia and Wolbachia are susceptible
to doxycycline and rifampicin, although these bacteria
show heterogeneous susceptibility to quinolone compounds
[21–23] (Table 1). Antibiotic susceptibility testing of
Coxiella burnetii showed that amikacin and amoxicillin
were not effective, whereas co-trimoxazole, rifampicin,
doxycycline, clarithromycin and quinolones were all bacte-
riostatic [24,25]. There is heterogeneity in susceptibility to
erythromycin among the strains tested [25,26]. Strains of
Brucella spp. also show heterogeneous susceptibility to ery-
thromycin but are susceptible to almost all antibiotics except
chloramphenicol, co-trimoxazole and ␤-lactams [27,28].
4. Methods for whole genome sequence analysis
Total numbers of bacterial genomes used in this study
are described in Fig. 2, which represents a phylogenetic
tree based on 16S sequence comparison. The target genes
involved in antibiotic resistance were retrieved from avail-
able genomes at the Kyoto Encyclopedia of Genes and
Genomes (KEGG) (http://www.genome.jp/kegg/) database.
The nucleotide sequences of target genes and/or amino acid
sequences were compared and aligned using the ClustalW
program (http://www.ebi.ac.uk/clustalw/) to examine pos-
sible mutations known to be associated with antibiotic
resistance. The keywords used for the in silico genome
study were: efflux; multidrug; ABC transporters; MFS;
RND; MATE; cat gene; tet gene; aminoglycoside-modifying
enzymes; chloramphenicol; folate (folA, folP); 23S; L4;
L22; 16S; erm gene; gyrA and gyrB gene; macrolide;
aminoglycoside; tetracycline; beta-lactam; fluoroquinolone;
rifampin; trimethoprim; and sulfamethoxazole.
5. Mode of action and mechanisms of antibiotic
resistance: in silico genomic analysis and study of
natural and in vitro mutants
Most antimicrobial agents used for the treatment of
bacterial infections can be categorised according to their
main mechanism of action. There are different modes of
Table 2
Candidate genes involved in antibiotic resistance
Antibiotic Candidate genes
Macrolides erm, mef, msr, 23S rRNA, rplD (L4
r-protein), rplV (L22 r-protein)
Tetracyclines 16S rRNA, tet gene, rpsL (S12 r-protein),
rpsG (S7r-protein)
Aminoglycosides 16S rRNA, rpsL (S12 r-protein),
aminoglycoside-modifying enzymes
Quinolones gyrA, gyrB, parC, parE
Rifampicin rpoB
Chloramphenicol cat, 23S rRNA
␤-Lactams mecA
Trimethoprim folA
Sulfamethoxazole folP
erm, erythromycin ribosome methylation; mef, macrolide efflux; msr,
methionine sulfoxide reductase; r-protein, ribosomal protein; tet, tetracy-
cline resistance protein; gyrA, DNA gyrase subunit A; gyrB, DNA gyrase
subunit B; parC, DNA topoisomerase IV subunit A; parE, DNA topoi-
somerase IV subunit B; rpoB, DNA-directed RNA polymerase subunit ␤;
cat, chloramphenicol acetyltransferase; mecA, penicillin-binding protein 2’;
folA, dihydrofolate reductase; folP, dihydropteroate synthase.
action for different antibiotics, including inhibition of protein
synthesis, interference with cell wall synthesis, interference
with nucleic acid synthesis and inhibition of metabolic
pathways. Candidate genes involved in the mechanisms of
antibiotic resistance are described in Table 2 and Fig. 3.
5.1. Inhibition of protein synthesis is the main
mechanism of action for macrolides, lincosamides,
chloramphenicol, aminoglycosides and tetracyclines
5.1.1. Macrolide–lincosamide–streptogramin (MLS)
antibiotics
The MLS antibiotics are an important group of translation
inhibitors that act on the 50S ribosomes [29]. The MLS group
was defined on the basis of cross-resistance patterns, which
showed that these drugs acted on the peptidyl transferase
centre of the 50S subunit. Binding of these drugs was found
to involve domains II and V of the 23S rRNA [4,5].
Intrinsic resistance to MLSB (macrolide–lincosamide–
streptogramin B) antibiotics in bacteria is generally due
to low permeability of the outer membrane to these
hydrophobic compounds [2]. Three different mechanisms
of acquired MLS resistance have been found in bacteria
[30–32]. The first described mechanism was a result of
post-transcriptional modifications of the 23S rRNA by
adenine-N6 -methyltransferase. Ribosomal target modifi-
cation confers cross-resistance to MLSB antibiotics and
remains the most frequent mechanism of resistance. The
genes encoding these methylases have been termed erm
(erythromycin ribosome methylation) [2,33,34]. In our study,
erm genes were not found in the genome of intracellular
bacteria.
Another mechanism of resistance is active drug efflux
mediated by the membrane-bound efflux protein encoded
by mef(A), which confers resistance only to 14- and
 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220 211

Fig. 3. (a) Action of macrolides on the peptidyl-tRNA molecule during elongation, resulting inhibition of protein synthesis. (b) Candidate genes for macrolide,
chloramphenicol, tetracycline and aminoglycoside resistance. (c) Secondary structure of Escherichia coli 23S rRNA showing the hairpin 35 in domain II and
part of domain V. The nucleotides in domain II and domain V whose mutations cause resistance to macrolides are shown by green dots. (d) Candidate genes
(gyrA, gyrB) for fluoroquinolones resistance. (e) Structure of RNA polymerase showing the ␤ subunit, the binding site of the antibiotic rifampicin. Rifampicin
inhibits DNA-dependent RNA polymerase by binding to the ␤ subunit encoded by the rpoB gene.

15-membered macrolides [2,35,36]. By in silico genome
analysis, macrolide-specific efflux proteins (e.g. MacA
and MacB) were identified in the Bartonella genome.
In the Escherichia coli genome, ybjYZ were suspected
to be genes for ABC drug efflux transporters and were
renamed macAB (macrolide-specific ABC-type efflux
carrier) [37,38]. Plasmids carrying both the macA and macB
genes conferred resistance against macrolides composed of
14- and 15-membered compounds, but conferred only weak
resistance against 16-membered compounds.
Another mechanism of resistance is mutation of the
bacterial 23S rRNA, or mutations, insertions or deletions
in ribosomal protein genes [34,39] (Fig. 3b). Mutations in
the 23S rRNA at position A2058 and/or A2059 remain the
most common and confer the highest levels of macrolide
resistance. A lower level of drug resistance is provided by
mutations at positions 2057, 2452 and 2611 of the 23S
rRNA [34] (Fig. 3c). Mutations in the single copies of genes
encoding the L4 and L22 ribosomal proteins have also been
implicated in macrolide resistance [39,40]. Amino acids
212 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220
59–90 and 85–87 in the L4 and L22 ribosomal proteins,
respectively, have been reported to be important mutational
regions for macrolide resistance [39,41–43].
Heterogeneity in susceptibility to erythromycin has
been shown among F. tularensis subsp. holarctica, with
biovar I being erythromycin sensitive and biovar II being
erythromycin resistant [19]. The molecular mechanisms of
resistance to erythromycin are not known, but alignment of
domain V of the 23S rRNA using in silico methods showed
an A → C transition at position 2059 (E. coli numbering)
in one F. tularensis genome (F. tularensis subsp. holarctica
LVS), which could explain the heterogeneity in susceptibility
to erythromycin for F. tularensis strains (Fig. 4). Recently,
we reported one natural mutation (A2059G) in the 23S rRNA
gene (Table 3) in a Bartonella henselae isolate from a lymph
node from a patient with cat-scratch disease, suggesting
that naturally occurring erythromycin-resistant strains may
infect humans.
A study by Branger et al. [44] confirmed that macrolides
and telithromycin lacked antimicrobial activity against
Ehrlichia [21–23,45]. They reported numerous specific
mutations in nucleotides known to confer resistance to
macrolides in the Ehrlichia chaffeensis 23S rRNA gene
(e.g. T754G, G2057A, A2059G and C2611T) [44]. Wol-
bachia pipientis, a closely related organism, also possesses
mutations T754A, A2058G, A2059C and C2611G, which
could explain the intrinsic resistance of this bacterium to
macrolides [46] (Table 3).
Recently, we found three amino acid differences in
the highly conserved region at the C terminus of the L22
ribosomal protein between typhus group (TG) rickettsiae
and spotted fever group (SFG) rickettsiae [47], which may
explain the heterogeneity in susceptibility to erythromycin
between these two subgroups. Rickettsia typhi and Rickettsia
prowazekii showed two amino acid changes at positions
83 and 84 (Streptococcus pneumoniae numbering) and a
single amino acid change at position 89 compared with the
seven SFG rickettsial strains [47]. The three amino acid
differences found between the two subgroups of rickettsiae
were located in a highly conserved region of the L22
protein. In E. coli, deletion of three amino acids in this
conserved region (Met82 -Lys83 -Arg84 ) conferred resistance
to erythromycin [48]. Similarly, amino acid substitutions
as well as insertions or deletions within the region between
amino acid positions 80 and 94 have been reported in
in vitro mutants of Haemophilus influenzae resistant to
macrolide compounds [49]. Finally, we found a macrolide
2 -phosphotransferase-like protein in the genome of T.
whipplei Twist.
5.1.1.1. In vitro mutants. In Bartonella spp., differ-
ent mechanisms of erythromycin resistance have been
reported using in vitro studies. We demonstrated that the
fully erythromycin-resistant strain of Bartonella quintana
obtained after 16 passages in vitro harboured a 27-base repeat
insertion in ribosomal protein L4, resulting in an insertion
of nine repeated amino acids between amino acids R71 and
A72 in the highly conserved region of the protein [50].
Recently, we reported various changes in the 23S rRNA
gene and the L4 ribosomal protein for the B. henselae strain
Marseille as well as other B. henselae isolates [51]. Most of
the mutations in the 23S rRNA gene (e.g. A2058G, A2058C
and C2611T) were previously reported to confer ery-
thromycin resistance in other bacteria as well [34,39,44,52].
We found amino acid mutations at two different posi-
tions (G71R and H75Y) in ribosomal protein L4 among
erythromycin-resistant strains of B. henselae [51] (Table 4).
The A2058G mutation in the erythromycin-resistant strain of
Bartonella bacilliformis was also reported by our team [53].
5.1.2. Chloramphenicol
Chloramphenicol is a bacteriostatic antimicrobial agent
that is effective against a wide variety of microorganisms.
Chloramphenicol interferes with microbial protein synthesis
by binding to the 50S ribosomal subunit and inhibiting the
peptidyltransferase step in protein synthesis [2].
There are three known mechanisms of resistance to
chloramphenicol: reduced membrane permeability; muta-
tion of the 23S ribosomal subunit; and elaboration of
chloramphenicol acetyltransferase. Mutations in 23S rRNA
have been previously reported in chloramphenicol-resistant
strains of E. coli and Ehrlichia [44].
High-level resistance to chloramphenicol is conferred
by the cat gene, which encodes an enzyme called chloram-
phenicol acetyltransferase that inactivates chloramphenicol
[54,55]. This enzyme is usually encoded on a plasmid and
can be transferred along with genes conferring resistance to
a number of other antibiotics [55]. Whole genome analysis
data showed the presence of a cat gene in the genome of
Bartonella.
5.1.3. Aminoglycosides
Aminoglycosides kill bacteria by inhibiting protein
synthesis via binding to the 16S rRNA and disrupting
the integrity of the bacterial cell membrane [56,57]. The
most frequently encountered mechanism of resistance
to aminoglycosides is their structural modification by
specific enzymes produced by resistant organisms. The three
classes of such aminoglycoside-modifying enzymes are:
(1) aminoglycoside nucleotidyltransferases, which transfer
nucleotide triphosphates; (2) aminoglycoside acetyltrans-
ferases, which transfer the acetyl group from acetyl-CoA;
and (3) aminoglycoside phosphotransferases, which transfer
the phosphoryl group from ATP [58–61].
It has previously been reported that the genome of
Rickettsia conorii contains one gene encoding a protein
similar to an aminoglycoside 3 -phosphotransferase, and
there is a streptomycin resistance protein homologue in the
genome of Rickettsia felis; on the other hand, the genomes of
R. typhi and R. prowazekii do not contain these genes [62]. A
predicted aminoglycoside phosphotransferase is also present
in the genome of Wolbachia Bma. In silico data showed the
 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220 213

Fig. 4. Alignment of 23S rRNA (domain V) sequences of different intracellular bacteria showing changes at position 2059 for Francisella tularensis subsp.
holarctica LVS (A2059C) and for Ehrlichia, Neorickettsia and Wolbachia spp. (A2059G).

presence of a probable aminoglycoside efflux pump (AcrD,
acriflavine resistance protein D) in the genome of Ehrlichia
ruminantium str. Welgevonden (France) and E. ruminantium
str. Gardel. Aminoglycoside N(6 )-acetyltransferase (aacA4)
was found in the genome of C. burnetii.
Other mechanisms of resistance include alteration of the
30S ribosomal subunit target by mutation (mutation in 16S
rRNA gene) (Fig. 3b), methylation of the aminoglycoside-
binding site, and reduction of the intracellular concentration
of aminoglycosides by changes in outer membrane per-
meability, decreased inner membrane transport and active
efflux [63–69].
Previous studies indicated that aminoglycosides could
not diffuse passively through the eukaryotic cell membrane
because of their large size and negative charge. Cellular
uptake of this class of antibiotics corresponded to an active
mechanism of pinocytosis by the eukaryotic cell, explaining
the slow intracellular accumulation of these drugs [45,70].
5.1.4. Tetracyclines
Tetracyclines are broad-spectrum antimicrobial agents
with activity against a broad range of pathogenic bacteria,
including intracellular bacteria [71,72]. Tetracycline is
thought to inhibit the growth of bacteria by entering the
bacterial cell, binding to ribosomes and inhibiting protein
synthesis [73]. Several studies have found a single, high-
affinity binding site for tetracyclines in the ribosomal 30S
subunit [74,75] (Fig. 3b).
In most species, resistance to tetracycline is conferred
by genes with two main modes of action. The first group of
genes encodes efflux systems that transport the drug from
the inside to the outside of the bacterial cell; the second
214 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220

Table 3
Genome analysis data and natural mutations in candidate genes for antibiotic resistance in intracellular bacteria
Intracellular bacteria Antibiotic class and mechanism of resistance

MAC FQa AMG BL RIF (rpoB)a CHL

23S rRNA L22 ribosomal protein gyrA parC
Bartonella henselae A2059G Ser-83 → Ala
Bartonella quintana Ser-83 → Ala
Bartonella bacilliformis Ser-83 → Ala MBL
Rickettsia spp. Triple AA differences APH BLA Phe-973 → Leu
Francisella tularensis A2059C BLA
Ehrlichia chaffeensis T754G Ser-83 → Ala MBL G2057A
G2057A
A2059G
C2611T
Wolbachia pipientis T754A MBL
A2058G
A2059C
C2611G
Tropheryma whipplei Ser-83 → Ala Ser-96 → Ala
Coxiella burnetii AAC MBL
Brucella suis MBL
MAC, macrolides; FQ, fluoroquinolones; AMG, aminoglycosides; BL, ␤-lactams; RIF, rifampicin; CHL, chloramphenicol; AA, amino acid; APH, aminogly-
coside phosphotransferase; AAC, aminoglycoside acetyltransferase; MBL, metallo-␤-lactamase; BLA, ␤-lactamase.
a Amino acid changes resulting from the gene mutation are given.

group encodes ribosomal protection proteins, which remove
tetracycline from the ribosome [72,76]. All of the tet efflux
genes encode membrane-associated proteins that export
tetracycline from the cell. These tetracycline resistance
determinants are often associated with transmissible genetic
elements including plasmids, transposons and integrons [72].
Genome data analysis revealed the presence of a tetracy-
cline resistance protein (Tet) in the genome of B. quintana,
most F. tularensis strains and Brucella ovis. Among several
tetracycline resistance determinants (TetA, TetB and TetC),
the genome of Brucella melitensis biovar Abortus possessed
the tetracycline resistance protein TetB. These proteins
generally interact with the ribosome and, as a result, protein
synthesis is unaffected by the presence of the antibiotics [2].
5.2. The cell wall can be affected by drugs that prevent
the production of new cell walls, leading to cell lysis and
death; β-lactam drugs such as penicillins,
cephalosporins and carbapenems all interfere with cell
wall production
5.2.1. β-Lactam antibiotics
␤-Lactam antibiotics are among the most commonly
used antimicrobial agents. They interfere with the final stage

Table 4
Molecular mechanisms of resistance in intracellular bacteria selected in vitro for different classes of antibiotics
Intracellular bacteria Antibiotic class and mechanism of resistance

MAC FQ (gyrA)a RIF (rpoB)a SMX (dhps)a

23S rRNA L4 ribosomal protein

Bartonella henselae A2058G G71R
A2058C H75Y
C2611T
Bartonella quintana Insertion GRARHSSAR
Bartonella bacilliformis A2058G Asp-87 → Asn Ser-531 → Phe
Rickettsia spp. Leu-151 → Phe
Phe-201 → Leu
Val-271 → Ile
Arg-546 → Lys
Francisella tularensis Thr-83 → Ile
Coxiella burnetii Glu-87 → Gly
Tropheryma whipplei Val-57 → Ile
Thr-102 → Pro
Leu-162 → Ile
MAC, macrolides; FQ, fluoroquinolones; RIF, rifampicin; SMX, sulfamethoxazole.
a Amino acid changes resulting from the gene mutation are given.
 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220
of cell wall synthesis by inhibiting the bacterial enzymes
transpeptidases and carboxypeptidases that catalyse the reac-
tions of peptidoglycan synthesis [77,78]. These enzymes,
commonly called penicillin-binding proteins (PBPs),
cross-link the peptidoglycan polymers. Peptidoglycan is an
essential component of the bacterial cell wall, and inhibition
of PBPs causes bacteriolysis by creating a wall unable to
withstand osmotic forces [77,79].
The greatest single cause of resistance to ␤-lactam antibi-
otics is antibiotic-inactivating enzymes, the ␤-lactamases,
which efficiently catalyse irreversible hydrolysis of the
amide bond of the ␤-lactam ring resulting in biologically
inactive products [80]. Over 250 ␤-lactamases have been
described, varying in their substrate profiles, inhibition
profiles, molecular mass, isoelectric point, amino acid
sequence and molecular structure [77,81,82]. Genes encod-
ing ␤-lactamases can be localised either on plasmids or
on the bacterial chromosome and are found both among
Gram-negative and Gram-positive organisms [2].
Whole genome analysis showed that most Francisella
strains possessed a blaA (␤-lactamase class A) gene and
AmpG protein. ␤-Lactamases of Amber’s class A are the
most commonly found in bacteria resistant to ␤-lactam
antibiotics [83]. The AmpG protein is an integral membrane
protein that functions as a peptidoglycan-specific permease
and can be used to transport new drugs mimicking the
murein recycled compounds into the cytoplasm [84].
Metallo-␤-lactamase family proteins were found in the
genomes of most of the intracellular bacteria used in this
study (Table 3).
Five specific open-reading frames (ORFs) related to
antibiotic resistance have been previously identified in
the genome of R. felis, including a class C ␤-lactamase, a
penicillin acylase homologue and an ABC-type multidrug
transporter system [85]. Interestingly, a previous study
showed the presence of two genes encoding ␤-lactamases in
the genome of R. conorii and none in the genome of R. typhi
and R. prowazekii, which possessed PBPs and ampG genes
instead [63].
5.3. Nucleic acid synthesis can be interrupted by several
mechanisms
5.3.1. Fluoroquinolones
Quinolones or fluoroquinolones are among the most
important antibacterial drugs and are used extensively for
the treatment of bacterial infections both in human and
veterinary medicine [86].
Fluoroquinolones exert their antibacterial effects by
inhibition of certain bacterial topoisomerase enzymes,
namely DNA gyrase and topoisomerase IV. DNA gyrase and
topoisomerase IV are heterotetrameric proteins composed
of two subunits, designated A and B [87–90]. The genes
encoding the A and B subunits are referred to as gyrA and
gyrB (DNA gyrase) or parC and parE (DNA topoisomerase
IV), respectively (Table 2; Fig. 3d). DNA gyrase is the only
215
enzyme that can affect supercoiling of DNA, and inhibition
of this activity by fluoroquinolones is associated with rapid
killing of the bacterial cell [2,91–93].
Alterations in target enzymes appear to be the most dom-
inant factors in expression of resistance to quinolones [92].
A small region from codon 67 to 106 of gyrA in E. coli was
designated the ‘quinolone resistance-determining region’
(QRDR) [2] and variations in this region were found in
species with natural resistance to fluoroquinolones [94,95].
Analysis of the T. whipplei genome allowed the identifi-
cation of the gyrA and parC gene encoding the ␣ subunit of
the natural fluoroquinolone targets DNA gyrase and topoiso-
merase IV, respectively [16]. Heterogeneity in susceptibility
to fluoroquinolones in T. whipplei [16] was found to be
associated with mutations in the DNA gyrase gene. In the T.
whipplei GyrA and ParC sequences, alanine residues were
found at positions 81 and 96, respectively, corresponding
to a serine at position 83 in E. coli GyrA [96] and a serine
at position 80 in E. coli ParC [97], respectively (Table 3).
GyrA-mediated natural resistance to fluoroquinolones has
also been described in Mycobacterium spp., which are
closely phylogenetically related to T. whipplei. In silico
genome analysis revealed a natural mutation at position 83
of the QRDR region (Ser-83 → Ala) of the GyrA protein
for three Bartonella species (Table 3). Many examples exist
demonstrating that species naturally bearing a serine residue
at position 83 of GyrA protein are usually susceptible to
fluoroquinolones, whereas the presence of an alanine at this
critical position corresponds to natural resistance to these
antibiotics [98–101]. Similarly, Maurin et al. [102] observed
that a serine residue at position 83 of the GyrA protein in
susceptible species of Ehrlichia is replaced by an alanine
residue in fluoroquinolone-resistant species.
5.3.1.1. In vitro mutants. Resistance to fluoroquinolones
has been described in some strains of C. burnetii and it was
shown that the mechanism involved two distinct nucleotide
mutations in the GyrA protein (Glu-87 → Gly and Glu-
87 → Lys) [103,104] (Table 4). An amino acid change
(Asp-87 → Asn) in its GyrA has also been reported recently
in a ciprofloxacin-resistant strain of B. bacilliformis (Table 4).
5.3.2. Rifampicin
The molecular mechanism of rifampicin activity involves
inhibition of DNA-dependent RNA polymerase. This enzyme
is a complex oligomer composed of four different subunits (␣,
␤, ␤ and ␴ encoded by rpoA, rpoB, rpoC and rpoD, respec-
tively) (Fig. 3e). Rifampicin binds to the ␤ subunit of RNA
polymerase and results in transcription inhibition [105,106].
Resistance to rifampicin is primarily caused by mutations
in the rpoB gene. In the majority of rifampicin-resistant
isolates, mutations occurred within an 81-bp hotspot region
(the rifampicin resistance-determining region (RRDR),
codons 507–533 according to E. coli numbering) in the
rpoB gene [4,107]. Previously, Rolain et al. [19] found that
susceptibility to rifampicin varied, with R. prowazekii, R.
216 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220

typhi, Rickettsia canada, Rickettsia bellii and most SFG The target for sulphonamide action is dihydropteroate
rickettsiae being susceptible to rifampicin, whilst the Rick- synthase (DHPS), which catalyses the condensation of
ettsia massiliae subgroup (R. massiliae, Rickettsia montana, para-aminobenzoic acid with 7,8-dihydro-6-hydroxy-
Rickettsia rhipicephalus and Rickettsia aeschlimannii) methylopterine pyrophosphate to form 7,8-dihydropteroate
were more resistant to rifampicin. Drancourt and Raoult [115–118]. Sulphonamide resistance is commonly mediated
[108] investigated the genetic basis for natural rifampicin by the presence of alternative drug-resistant forms of DHPS.
resistance in representatives of the TG and the two SFG Chromosomal mutations in the dhps gene that confer
subgroups of rickettsiae by sequence analysis of the rpoB resistance to sulphonamides have also been identified in a
gene. They found a single point mutation resulting in a number of bacteria [116,119].
Phe → Leu change at position 973 of the R. conorii rpoB Mutation in folA and folP (structural genes for DHFR and
sequence (Table 3). This single point mutation, which DHPS, respectively) could confer resistance to trimethoprim
appeared to be specific for the naturally rifampicin-resistant and sulfamethoxazole, respectively. Interestingly, Rickettsia
subgroup, was not previously implicated in rifampicin spp. are resistant to co-trimoxazole and it has been found
resistance in other bacteria. Resistance to rifampicin can that the folP and folA genes are absent in most Rickettsia
also be due to expression of an efflux system [109,110]. spp. Coxiella burnetii, which is naturally susceptible to
co-trimoxazole compounds, showed both folP and folA genes
in its genome. Interestingly, T. whipplei is susceptible to
5.3.2.1. In vitro mutants. Amino acid substitutions in the
co-trimoxazole, although only the folP gene is present in the
RNA polymerase and rpoB point mutations have been
Tropheryma genome. A recent study [120] demonstrated that
demonstrated following in vitro selection of rifampicin-
the MICs against the two strains of T. whipplei ranged from
resistant R. prowazekii [111] and R. typhi [112]. Troyer et al.
0.5 mg/L to 1 mg/L for sulfadiazine compared with 0.5 mg/L
[112] reported the detection of a rifampicin-resistant strain of
for sulfamethoxazole, leading the authors to suggest that
R. typhi (Ethiopian). The basis of this resistance was inves-
sulfadiazine was as effective as sulfamethoxazole in vitro.
tigated by sequencing and mapping point mutations in the
rpoB gene of the mutant and then comparing the sequences
5.4.1.1. In vitro study. We developed a new method to study
of wild-type and rifampicin-resistant R. typhi rpoB genes. A
antibiotic susceptibility in fastidious bacteria such as T. whip-
total of eight nucleotide substitutions occurred, three of which
plei using E. coli gene complementation. In the genome of T.
resulted in amino acid substitutions in the mutant strain:
whipplei, a typical DHPS-encoding gene, the target gene for
leucine for phenylalanine at residue 151, phenylalanine for
sulfamethoxazole, is not found as an individual ORF. DNA
leucine at residue 201 and valine for isoleucine at residue
sequencing of two samples (before and after failure) from
271 [112] (Table 4). In another study by Rachek et al., com-
a patient with clinically acquired resistance to trimetho-
parison of the rpoB sequences from the rifampicin-sensitive
prim/sulfamethoxazole, using specific oligonucleotide
R. prowazekii Madrid E strain and a rifampicin-resistant
primers for the candidate gene folP, showed three amino
mutant identified a single point mutation that resulted in an
acid changes [121]. Gene complementation in E. coli showed
arginine-to-lysine change at position 546 of the rpoB gene
that the mutated sequence was associated with resistance.
[111].
A recently reported rifampicin-resistant strain of B.
bacilliformis from our group showed a mutation at serine
6. Efflux pumps
531 (Ser → Phe) in the RRDR of the rpoB gene [53]
(Table 4). This 531 site is one of the most frequent sites
Efflux pumps are present in all living cells. They partici-
of mutation, also conferring rifampicin resistance in other
pate in the detoxification process, expelling various harmful
bacterial species [106].

5.4. Folate synthesis, which is necessary for DNA

replication, is blocked by sulphonamides and
trimethoprim

5.4.1. Trimethoprim and sulphonamides

Trimethoprim is an analogue of dihydrofolic acid, an
essential component in the synthesis of amino acids and
nucleotides, which competitively inhibits the enzyme
dihydrofolate reductase (DHFR). Resistance can be caused
by a number of mechanisms, including overproduction of
host DHFR, mutations in the structural gene for DHFR
and acquisition of a foreign gene (dfr) encoding a resistant Fig. 5. Correlation between genome size of intracellular bacteria and number
DHFR enzyme [2,113,114]. of ATP-binding cassette (ABC) transporters present in the genome.
 S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220
compounds and xenobiotics [122,123]. Bacterial drug efflux
pumps are currently classified into five families [124–130]:
(i) the ATP-binding cassette (ABC) superfamily; (ii) MFS
(major facilitator superfamily) transporters; (iii) the RND
(resistance–nodulation–cell division) superfamily; (iv) the
SMR (small multidrug resistance) family; and (v) the MATE
(multidrug and toxic compound extrusion) family.
6.1. In silico genome analysis
Whole genome analysis using an in silico method showed
that the number of genes encoding ABC transporters in
intracellular bacteria varied from four (R. prowazekii, E.
ruminantium Welgevonden France and E. ruminantium
Gardel) to 277 (Brucella suis). We found a correlation
between the numbers of encoded ABC transporters and
genome size, which is depicted in Fig. 5. Genome size
and number of ABC transporters in different intracellular
bacteria are given in Supplementary Data 1.
Several of the efflux systems found among intracellular
bacteria are given in Supplementary Data 2. Efflux systems
including pH adaptation potassium efflux system proteins
(PhaAB, PhaC, PhaD, PhaE, PhaF and PhaG) have been
found in multiple different Bartonella spp. The genome of T.
whipplei Twist contains the multidrug efflux protein QacA
and a cation efflux protein. The plasmid-encoded multidrug
resistance gene, qacA, from Staphylococcus aureus mediates
resistance to a number of classes of antimicrobial organic
cations, including intercalating dyes, quaternary ammonium
compounds, diamidines and biguanidines [70,76]. Interest-
ingly, T. whipplei has been shown to be resistant to several
antiseptics, including glutaraldehyde and peracetic acid
[131].
In the genome of F. tularensis we found outer membrane
efflux proteins along with other efflux systems. The OEP
(outer membrane efflux protein) family forms trimeric
channels allowing export of a variety of substrates in
Gram-negative bacteria [132].
The glutathione-regulated potassium efflux system
protein, KefB, is present in the genome of all Rickettsia spp.
and is also known as K+ /H+ antiporter. It is localised to the
inner membrane of the cell and facilitates potassium efflux
[133].
7. Concluding remarks and perspectives
The emergence and spread of antimicrobial resistance
determinants continues to challenge our ability to treat
serious infections. The past two decades have produced
substantial research into the mechanisms by which bacteria
develop and disperse resistance determinants, although there
is still much to be learned. In silico genome analysis will
help to predict possible molecular mechanisms of resistance
among intracellular bacteria. Studying microbial genomes
will also aid in the discovery process by identifying all bacte-
217
rial gene products that may be involved in antibiotic transport
and efflux from bacterial cells. Among intracellular bacteria,
the spread of antibiotic resistance is mainly due to vertical
gene transfer, which is also observed in M. tuberculosis. This
group of organisms could be an interesting paradigm to iden-
tify different resistance determinants using whole genome
analysis. Microarray studies could also be used to determine
antibiotic resistance genes in intracellular bacteria. The
study of natural mutants and in vitro resistant mutants will
additionally help in the understanding of the efficacy of
different classes of antibiotics among intracellular bacteria.
We developed a new method to study antibiotic resistance
in fastidious bacteria (e.g. T. whipplei) using E. coli gene
complementation. Transformation of intracellular bacteria
is one of the difficulties we face in working with these
microorganisms. This complementation approach could be
used with other intracellular bacteria for further study and
could open a door to the identification and demonstration
of resistance determinants among these bacteria. Genomic
studies will further clarify how resistance to novel classes
of antibiotics arises, in addition to the fitness costs to the
organism that result from resistance.
Funding: No funding sources.
Competing interests: None declared.
Ethical approval: Not required.
Appendix A. Supplementary data
Supplementary data associated with this arti-
cle can be found, in the online version, at
doi:10.1016/j.ijantimicag.2008.03.017.
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