International Journal of Antimicrobial Agents 32 (2008) 207–220
Review
A bioinformatic approach to understanding antibiotic resistance inintracellular 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
bstract
Intracellular bacteria survive within eukaryotic host cells and are difficult to kill with certain antibiotics. As a result, antibiotic resistancen intracellular bacteria is becoming commonplace in healthcare institutions. Owing to the lack of methods available for transforming theseacteria, we evaluated the mechanisms of resistance using molecular methods and in silico genome analysis. The objective of this reviewas to understand the molecular mechanisms of antibiotic resistance through in silico comparisons of the genomes of obligate and facultative
ntracellular bacteria. The available data on in vitro mutants reported for intracellular bacteria were also reviewed. These genomic data werenalysed to find natural mutations in known target genes involved in antibiotic resistance and to look for the presence or absence of differ-nt resistance determinants. Our analysis revealed the presence of tetracycline resistance protein (Tet) in Bartonella quintana, Francisellaularensis and Brucella ovis; moreover, most of the Francisella strains possessed the blaA gene, AmpG protein and metallo-�-lactamase familyrotein. The presence or absence of folP (dihydropteroate synthase) and folA (dihydrofolate reductase) genes in the genome could explainatural resistance to co-trimoxazole. Finally, multiple genes encoding different efflux pumps were studied. This in silico approach was an
ffective method for understanding the mechanisms of antibiotic resistance in intracellular bacteria. The whole genome sequence analysis willelp to predict several important phenotypic characteristics, in particular resistance to different antibiotics. In the future, stable mutants shoulde 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.
Intracellular bacteria are defined by their capacity tourvive and live inside eukaryotic host cells. As a result,hey have developed diverse strategies to survive within thisompartment. These bacteria are responsible for enormousorbidity and mortality worldwide and are difficult to killith certain antibiotics. The barrier to antibiotic treatment of
bligate intracellular bacteria is the difference between theocalisation of antibiotics within the cellular compartmentsf infected cells and the localisation of the bacteria [1].
∗ Corresponding author. Present address: Unité des Rickettsies, CNRSMR 6020, IFR 48, Faculté de Médecine et de Pharmacie, Université de laéditerranée, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France.
Treating bacterial infections is increasingly complicatedy the ability of bacteria to develop resistance to differentntibiotics. Resistance to antibiotics can be caused by aariety of mechanisms: (i) the presence of an enzyme thatnactivates the antimicrobial agent; (ii) a mutation in the targetf the antimicrobial agent that reduces its binding capacity;iii) post-transcriptional and post-translational modificationf the target of the antimicrobial agent, which reduces itsinding capacity; (iv) reduced uptake of the antimicrobialgent; and (v) active efflux of the antimicrobial agent [2].
Bacteria can develop resistance to antibiotics throughwo genetic processes: (i) mutation and selection (verticalene transfer); and (ii) exchange of genes between strains
nd species (horizontal gene transfer). Among intracellularacteria, antibiotic resistance is primarily due to spontaneousutations or multiple mutations in the bacterial genome (i.e.
ertical gene transfer). To date, there is only one example of
of Chemotherapy. All rights reserved.
2 l of Antimicrobial Agents 32 (2008) 207–220
haItcamatdana
oabs
2a
atwwi
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Fig. 1. Lifestyles of intracellular bacteria: (1) bacterial escape into thecytosol after exit from the endosomal compartment (e.g. Rickettsia, Shigella,Listeria); (2) survival in non-fused phagosomes (e.g. Bartonella, Brucella,Lut
afRpsva
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08 S. Biswas et al. / International Journa
orizontal gene transfer conferring resistance to tetracyclinemong intracellular bacteria, namely Chlamydia suis [3].n Mycobacterium tuberculosis, the causative agent ofuberculosis, all of the drug resistance determinants arehromosomally encoded, arising exclusively through thecquisition and maintenance of spontaneous chromosomalutations in target or complementary genes [4]. Resistance-
ssociated point mutations, deletions or insertions in M.uberculosis have been previously described for all first-linerugs (e.g. isoniazid, rifampicin, pyrazinamide, ethambutolnd streptomycin) in addition to several second-line andewer drugs (e.g. ethionamide, fluoroquinolones, macrolidesnd nitroimidazopyrans) [5].
The objective of this review was to present an overviewf the molecular evidence for bacterial resistance tontimicrobial agents among intracellular bacteria using aioinformatic analysis of whole genome sequences and inilico analysis of target genes.
. Intracellular behaviour of bacteria and antibioticctivity
The intracellular localisation of some bacteria remainscritical point explaining the failure of some antibiotic
reatments in infected hosts. Parasites that multiply onlyithin eukaryotic cells are obligate intracellular pathogens,
hereas facultative intracellular pathogens can also multiply
n cell-free models [6].Four categories of mechanisms exist to explain the sur-
ival of intracellular bacteria: (a) survival in the cytoplasm
acim
able 1ntibiotic susceptibility results for various intracellular bacteria
ntracellular bacteria Antibiotic
ERY AMG TET
artonella spp. S S Sropheryma whipplei S S Srancisella tularensis S/R S S
ickettsia spp.Typhus group
R. prowazekii S R SR. typhi S R S
potted fever groupR. conorii subgroup
R. conorii, R. rickettsii R R S
R. massiliae subgroupR. massiliae, R. montanensis R R S
Ehrlichia spp.E. canis R R SE. chaffeensis R R SWolbachia spp. R R SCoxiella burnetii S/R R SBrucella spp. S/R S S
egionella); (3) segregation from the endolytic route and formation of anique inclusion vacuole (e.g. Chlamydia) and (4) survival by fusion withhe lysosome (e.g. Coxiella, Tropheryma, Francisella).
fter exit from an endosomal compartment with or withoutusion of the phagosomal vacuole with lysosomes (e.g.ickettsia, Shigella and Listeria); (b) survival in non-fusedhagosomes (e.g. Bartonella, Brucella and Legionella); (c)urvival in fused phagosomes (e.g. Chlamydia); and (d) sur-ival in fused phagolysosomes (e.g. Coxiella, Tropherymand Francisella) (Fig. 1).
Antibiotic activity against intracellular bacteria dependsn several factors, including pharmacodynamic and phar-acokinetic properties of antibiotics. First, in order to
e active, antibiotics must reach the infected cells inheir tissue compartments via the systemic route. Second,
ntibiotics need to reach and concentrate within intracellularompartments. The intracellular to extracellular ratio (C/E)s a very important factor and can be determined by several
ethods, including radiometric, fluorometric and chemical
echniques. For example, the fluoroquinolones accumulateithin phagocytes with a C/E ratio of 6:7 for granulo-
ytes, 3:4 for macrophages and ca. 2:1 for epithelial cells7–9]. Third, antibiotics should remain active within theargeted intracellular compartment, without inactivation byellular metabolism and/or deleterious effect of pH [6,10].ome antibiotics are more effective at neutral or basic pHalues (e.g. fluoroquinolone compounds) but others (e.g.ifampicin) are more effective at acidic pH values [6].
. Overview of natural antibiotic susceptibilitymong intracellular bacteria
The antibiotic susceptibility of facultative intracellularacteria can be assessed in cell-free systems using minimumnhibitory concentration (MIC) determination methods;or obligate intracellular bacteria, however, antibiotic
at
o
Fig. 2. Phylogenetic tree based on 16S sequences
imicrobial Agents 32 (2008) 207–220 209
usceptibility should be determined only in cell models.he choice of cell system depends on each pathogen, but
n general cell lines that are easy to obtain and grow aresed (e.g. Vero, L929 or MRC5 cells) [6]. The methods forvaluating antibiotic susceptibility vary with the nature ofhe intracellular pathogen [11]. One important techniques enumeration of viable intracellular microorganismscolony-forming unit or plaque assay) after various times ofntibiotic exposure compared with drug-free controls (e.g.ickettsia, Coxiella) [6]. Other methods of evaluation havelso been used, including determination of the percentagef infected cells, flow cytometry, immunofluorescence tech-iques, luciferase techniques and quantitative polymerasehain reaction (PCR) [7,12]. There are very few reports of
ntibiotic susceptibility testing among fastidious bacteria, ashe methods are time consuming and labour intensive.
Table 1 summarises the natural susceptibility to antibi-tics among intracellular bacteria. Bacteria of the genus
of intracellular bacteria used in this study.
2 l of Antimicrobial Agents 32 (2008) 207–220
B�m[taflsrcrTs
vts[CwdreBtc
4
atiaGTspsrsReLar
5rn
bm
Table 2Candidate genes involved in antibiotic resistance
artonella are susceptible to all antibiotics in vitro, including-lactams, aminoglycosides, chloramphenicol, tetracyclines,acrolides, rifampicin, fluoroquinolones and co-trimoxazole
13,14]. Tropheryma whipplei displays a homogeneous pat-ern of antibiotic susceptibility in axenic medium [15], withlmost all antibiotics showing at least some activity, exceptuoroquinolones [16]. Francisella tularensis strains areusceptible to streptomycin, gentamicin, doxycycline, chlo-amphenicol and quinolones but show heterogeneous sus-eptibility to erythromycin [17,18]. Rickettsia are naturallyesistant to �-lactams, aminoglycosides and co-trimoxazole.he typhus group is susceptible to erythromycin, whereas thepotted fever group is not [19,20] (Table 1).
In vitro antibiotic susceptibility studies have shown thatarious species of Ehrlichia and Wolbachia are susceptibleo doxycycline and rifampicin, although these bacteriahow heterogeneous susceptibility to quinolone compounds21–23] (Table 1). Antibiotic susceptibility testing ofoxiella burnetii showed that amikacin and amoxicillinere not effective, whereas co-trimoxazole, rifampicin,oxycycline, clarithromycin and quinolones were all bacte-iostatic [24,25]. There is heterogeneity in susceptibility torythromycin among the strains tested [25,26]. Strains ofrucella spp. also show heterogeneous susceptibility to ery-
hromycin but are susceptible to almost all antibiotics excepthloramphenicol, co-trimoxazole and �-lactams [27,28].
. Methods for whole genome sequence analysis
Total numbers of bacterial genomes used in this studyre described in Fig. 2, which represents a phylogeneticree based on 16S sequence comparison. The target genesnvolved in antibiotic resistance were retrieved from avail-ble genomes at the Kyoto Encyclopedia of Genes andenomes (KEGG) (http://www.genome.jp/kegg/) database.he nucleotide sequences of target genes and/or amino acidequences were compared and aligned using the ClustalWrogram (http://www.ebi.ac.uk/clustalw/) to examine pos-ible mutations known to be associated with antibioticesistance. The keywords used for the in silico genometudy were: efflux; multidrug; ABC transporters; MFS;ND; MATE; cat gene; tet gene; aminoglycoside-modifyingnzymes; chloramphenicol; folate (folA, folP); 23S; L4;22; 16S; erm gene; gyrA and gyrB gene; macrolide;minoglycoside; tetracycline; beta-lactam; fluoroquinolone;ifampin; trimethoprim; and sulfamethoxazole.
. Mode of action and mechanisms of antibioticesistance: in silico genomic analysis and study ofatural and in vitro mutants
Most antimicrobial agents used for the treatment ofacterial infections can be categorised according to theirain mechanism of action. There are different modes of
b
mb
ubunit B; parC, DNA topoisomerase IV subunit A; parE, DNA topoi-omerase IV subunit B; rpoB, DNA-directed RNA polymerase subunit �;at, chloramphenicol acetyltransferase; mecA, penicillin-binding protein 2’;olA, dihydrofolate reductase; folP, dihydropteroate synthase.
ction for different antibiotics, including inhibition of proteinynthesis, interference with cell wall synthesis, interferenceith nucleic acid synthesis and inhibition of metabolicathways. Candidate genes involved in the mechanisms ofntibiotic resistance are described in Table 2 and Fig. 3.
.1. Inhibition of protein synthesis is the mainechanism of action for macrolides, lincosamides,
The MLS antibiotics are an important group of translationnhibitors that act on the 50S ribosomes [29]. The MLS groupas defined on the basis of cross-resistance patterns, which
howed that these drugs acted on the peptidyl transferaseentre of the 50S subunit. Binding of these drugs was foundo involve domains II and V of the 23S rRNA [4,5].
Intrinsic resistance to MLSB (macrolide–lincosamide–treptogramin B) antibiotics in bacteria is generally dueo low permeability of the outer membrane to theseydrophobic compounds [2]. Three different mechanismsf acquired MLS resistance have been found in bacteria30–32]. The first described mechanism was a result ofost-transcriptional modifications of the 23S rRNA bydenine-N6-methyltransferase. Ribosomal target modifi-ation confers cross-resistance to MLSB antibiotics andemains the most frequent mechanism of resistance. Theenes encoding these methylases have been termed ermerythromycin ribosome methylation) [2,33,34]. In our study,rm genes were not found in the genome of intracellular
acteria.
Another mechanism of resistance is active drug effluxediated by the membrane-bound efflux protein encoded
y 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,c structup ions ca( ase showi ded by
1aaItrcg1r
bitmr
hloramphenicol, tetracycline and aminoglycoside resistance. (c) Secondaryart of domain V. The nucleotides in domain II and domain V whose mutatgyrA, gyrB) for fluoroquinolones resistance. (e) Structure of RNA polymernhibits DNA-dependent RNA polymerase by binding to the � subunit enco
5-membered macrolides [2,35,36]. By in silico genomenalysis, macrolide-specific efflux proteins (e.g. MacAnd MacB) were identified in the Bartonella genome.n the Escherichia coli genome, ybjYZ were suspectedo be genes for ABC drug efflux transporters and wereenamed macAB (macrolide-specific ABC-type efflux
arrier) [37,38]. Plasmids carrying both the macA and macBenes conferred resistance against macrolides composed of4- and 15-membered compounds, but conferred only weakesistance against 16-membered compounds.
mrei
re of Escherichia coli 23S rRNA showing the hairpin 35 in domain II anduse resistance to macrolides are shown by green dots. (d) Candidate genes
ing the � subunit, the binding site of the antibiotic rifampicin. Rifampicinthe rpoB gene.
Another mechanism of resistance is mutation of theacterial 23S rRNA, or mutations, insertions or deletionsn ribosomal protein genes [34,39] (Fig. 3b). Mutations inhe 23S rRNA at position A2058 and/or A2059 remain the
ost common and confer the highest levels of macrolideesistance. A lower level of drug resistance is provided by
utations at positions 2057, 2452 and 2611 of the 23S
RNA [34] (Fig. 3c). Mutations in the single copies of genesncoding the L4 and L22 ribosomal proteins have also beenmplicated in macrolide resistance [39,40]. Amino acids
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aEmm(bmcm
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5erfoi
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bp[cadB
5
stmtsc(nfat
Rst
12 S. Biswas et al. / International Journa
9–90 and 85–87 in the L4 and L22 ribosomal proteins,espectively, have been reported to be important mutationalegions for macrolide resistance [39,41–43].
Heterogeneity in susceptibility to erythromycin haseen shown among F. tularensis subsp. holarctica, withiovar I being erythromycin sensitive and biovar II beingrythromycin resistant [19]. The molecular mechanisms ofesistance to erythromycin are not known, but alignment ofomain V of the 23S rRNA using in silico methods showedn A → C transition at position 2059 (E. coli numbering)n one F. tularensis genome (F. tularensis subsp. holarcticaVS), which could explain the heterogeneity in susceptibilityo erythromycin for F. tularensis strains (Fig. 4). Recently,e reported one natural mutation (A2059G) in the 23S rRNAene (Table 3) in a Bartonella henselae isolate from a lymphode from a patient with cat-scratch disease, suggestinghat naturally occurring erythromycin-resistant strains maynfect humans.
A study by Branger et al. [44] confirmed that macrolidesnd telithromycin lacked antimicrobial activity againsthrlichia [21–23,45]. They reported numerous specificutations in nucleotides known to confer resistance toacrolides in the Ehrlichia chaffeensis 23S rRNA gene
e.g. T754G, G2057A, A2059G and C2611T) [44]. Wol-achia pipientis, a closely related organism, also possessesutations T754A, A2058G, A2059C and C2611G, which
ould explain the intrinsic resistance of this bacterium toacrolides [46] (Table 3).Recently, we found three amino acid differences in
he highly conserved region at the C terminus of the L22ibosomal protein between typhus group (TG) rickettsiaend spotted fever group (SFG) rickettsiae [47], which mayxplain the heterogeneity in susceptibility to erythromycinetween these two subgroups. Rickettsia typhi and Rickettsiarowazekii showed two amino acid changes at positions3 and 84 (Streptococcus pneumoniae numbering) and aingle amino acid change at position 89 compared with theeven SFG rickettsial strains [47]. The three amino acidifferences found between the two subgroups of rickettsiaeere located in a highly conserved region of the L22rotein. In E. coli, deletion of three amino acids in thisonserved region (Met82-Lys83-Arg84) conferred resistanceo erythromycin [48]. Similarly, amino acid substitutionss well as insertions or deletions within the region betweenmino acid positions 80 and 94 have been reported inn vitro mutants of Haemophilus influenzae resistant to
acrolide compounds [49]. Finally, we found a macrolide′-phosphotransferase-like protein in the genome of T.hipplei Twist.
.1.1.1. In vitro mutants. In Bartonella spp., differ-nt mechanisms of erythromycin resistance have been
eported using in vitro studies. We demonstrated that theully erythromycin-resistant strain of Bartonella quintanabtained after 16 passages in vitro harboured a 27-base repeatnsertion in ribosomal protein L4, resulting in an insertion
gRpi
imicrobial Agents 32 (2008) 207–220
f nine repeated amino acids between amino acids R71 and72 in the highly conserved region of the protein [50].Recently, we reported various changes in the 23S rRNA
ene and the L4 ribosomal protein for the B. henselae strainarseille as well as other B. henselae isolates [51]. Most of
he mutations in the 23S rRNA gene (e.g. A2058G, A2058Cnd C2611T) were previously reported to confer ery-hromycin resistance in other bacteria as well [34,39,44,52].
e found amino acid mutations at two different posi-ions (G71R and H75Y) in ribosomal protein L4 amongrythromycin-resistant strains of B. henselae [51] (Table 4).he A2058G mutation in the erythromycin-resistant strain ofartonella bacilliformis was also reported by our team [53].
.1.2. ChloramphenicolChloramphenicol is a bacteriostatic antimicrobial agent
hat is effective against a wide variety of microorganisms.hloramphenicol interferes with microbial protein synthesisy binding to the 50S ribosomal subunit and inhibiting theeptidyltransferase step in protein synthesis [2].
There are three known mechanisms of resistance tohloramphenicol: reduced membrane permeability; muta-ion of the 23S ribosomal subunit; and elaboration ofhloramphenicol acetyltransferase. Mutations in 23S rRNAave been previously reported in chloramphenicol-resistanttrains of E. coli and Ehrlichia [44].
High-level resistance to chloramphenicol is conferredy the cat gene, which encodes an enzyme called chloram-henicol acetyltransferase that inactivates chloramphenicol54,55]. This enzyme is usually encoded on a plasmid andan be transferred along with genes conferring resistance tonumber of other antibiotics [55]. Whole genome analysis
ata showed the presence of a cat gene in the genome ofartonella.
.1.3. AminoglycosidesAminoglycosides kill bacteria by inhibiting protein
ynthesis via binding to the 16S rRNA and disruptinghe integrity of the bacterial cell membrane [56,57]. The
ost frequently encountered mechanism of resistanceo aminoglycosides is their structural modification bypecific enzymes produced by resistant organisms. The threelasses of such aminoglycoside-modifying enzymes are:1) aminoglycoside nucleotidyltransferases, which transferucleotide triphosphates; (2) aminoglycoside acetyltrans-erases, which transfer the acetyl group from acetyl-CoA;nd (3) aminoglycoside phosphotransferases, which transferhe phosphoryl group from ATP [58–61].
It has previously been reported that the genome ofickettsia conorii contains one gene encoding a proteinimilar to an aminoglycoside 3′-phosphotransferase, andhere is a streptomycin resistance protein homologue in the
enome of Rickettsia felis; on the other hand, the genomes of. typhi and R. prowazekii do not contain these genes [62]. Aredicted aminoglycoside phosphotransferase is also presentn the genome of Wolbachia Bma. In silico data showed the
S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207–220 213
F llular bh pp. (A2
parsw
3rbome
nbu
mt
5
witbsas
ig. 4. Alignment of 23S rRNA (domain V) sequences of different intraceolarctica LVS (A2059C) and for Ehrlichia, Neorickettsia and Wolbachia s
resence of a probable aminoglycoside efflux pump (AcrD,criflavine resistance protein D) in the genome of Ehrlichiauminantium str. Welgevonden (France) and E. ruminantiumtr. Gardel. Aminoglycoside N(6′)-acetyltransferase (aacA4)as found in the genome of C. burnetii.Other mechanisms of resistance include alteration of the
0S ribosomal subunit target by mutation (mutation in 16SRNA gene) (Fig. 3b), methylation of the aminoglycoside-inding site, and reduction of the intracellular concentrationf aminoglycosides by changes in outer membrane per-eability, decreased inner membrane transport and active
fflux [63–69].
Previous studies indicated that aminoglycosides could
ot diffuse passively through the eukaryotic cell membraneecause of their large size and negative charge. Cellularptake of this class of antibiotics corresponded to an active
bgt
acteria showing changes at position 2059 for Francisella tularensis subsp.059G).
echanism of pinocytosis by the eukaryotic cell, explaininghe slow intracellular accumulation of these drugs [45,70].
.1.4. TetracyclinesTetracyclines are broad-spectrum antimicrobial agents
ith activity against a broad range of pathogenic bacteria,ncluding intracellular bacteria [71,72]. Tetracycline ishought to inhibit the growth of bacteria by entering theacterial cell, binding to ribosomes and inhibiting proteinynthesis [73]. Several studies have found a single, high-ffinity binding site for tetracyclines in the ribosomal 30Subunit [74,75] (Fig. 3b).
In most species, resistance to tetracycline is conferredy genes with two main modes of action. The first group ofenes encodes efflux systems that transport the drug fromhe 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 3Genome 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 → AlaBartonella quintana Ser-83 → AlaBartonella bacilliformis Ser-83 → Ala MBLRickettsia spp. Triple AA differences APH BLA Phe-973 → LeuFrancisella tularensis A2059C BLAEhrlichia chaffeensis T754G Ser-83 → Ala MBL G2057A
G2057AA2059GC2611T
Wolbachia pipientis T754A MBLA2058GA2059CC2611G
Tropheryma whipplei Ser-83 → Ala Ser-96 → AlaCoxiella burnetii AAC MBLBrucella suis MBL
M ctams;c metallo
gtgtde
cmttt
gs
5tdcw
TM
I
B
BBR
FCT
M
AC, macrolides; FQ, fluoroquinolones; AMG, aminoglycosides; BL, �-laoside phosphotransferase; AAC, aminoglycoside acetyltransferase; MBL,a Amino acid changes resulting from the gene mutation are given.
roup encodes ribosomal protection proteins, which removeetracycline from the ribosome [72,76]. All of the tet effluxenes encode membrane-associated proteins that exportetracycline from the cell. These tetracycline resistanceeterminants are often associated with transmissible geneticlements including plasmids, transposons and integrons [72].
Genome data analysis revealed the presence of a tetracy-line resistance protein (Tet) in the genome of B. quintana,
ost F. tularensis strains and Brucella ovis. Among several
etracycline resistance determinants (TetA, TetB and TetC),he genome of Brucella melitensis biovar Abortus possessedhe tetracycline resistance protein TetB. These proteins
5
u
able 4olecular mechanisms of resistance in intracellular bacteria selected in vitro for di
ntracellular bacteria Antibiotic class and mechanism of resistance
enerally interact with the ribosome and, as a result, proteinynthesis is unaffected by the presence of the antibiotics [2].
.2. The cell wall can be affected by drugs that preventhe production of new cell walls, leading to cell lysis andeath; β-lactam drugs such as penicillins,ephalosporins and carbapenems all interfere with cellall production
.2.1. β-Lactam antibiotics�-Lactam antibiotics are among the most commonly
sed antimicrobial agents. They interfere with the final stage
f cell wall synthesis by inhibiting the bacterial enzymesranspeptidases and carboxypeptidases that catalyse the reac-ions of peptidoglycan synthesis [77,78]. These enzymes,ommonly called penicillin-binding proteins (PBPs),ross-link the peptidoglycan polymers. Peptidoglycan is anssential component of the bacterial cell wall, and inhibitionf PBPs causes bacteriolysis by creating a wall unable toithstand osmotic forces [77,79].The greatest single cause of resistance to �-lactam antibi-
tics is antibiotic-inactivating enzymes, the �-lactamases,hich efficiently catalyse irreversible hydrolysis of the
mide bond of the �-lactam ring resulting in biologicallynactive products [80]. Over 250 �-lactamases have beenescribed, varying in their substrate profiles, inhibitionrofiles, molecular mass, isoelectric point, amino acidequence and molecular structure [77,81,82]. Genes encod-ng �-lactamases can be localised either on plasmids orn the bacterial chromosome and are found both amongram-negative and Gram-positive organisms [2].Whole genome analysis showed that most Francisella
trains possessed a blaA (�-lactamase class A) gene andmpG protein. �-Lactamases of Amber’s class A are theost commonly found in bacteria resistant to �-lactam
ntibiotics [83]. The AmpG protein is an integral membranerotein that functions as a peptidoglycan-specific permeasend can be used to transport new drugs mimicking theurein recycled compounds into the cytoplasm [84].Metallo-�-lactamase family proteins were found in the
enomes of most of the intracellular bacteria used in thistudy (Table 3).
Five specific open-reading frames (ORFs) related tontibiotic resistance have been previously identified inhe genome of R. felis, including a class C �-lactamase, aenicillin acylase homologue and an ABC-type multidrugransporter system [85]. Interestingly, a previous studyhowed the presence of two genes encoding �-lactamases inhe genome of R. conorii and none in the genome of R. typhind R. prowazekii, which possessed PBPs and ampG genesnstead [63].
.3. Nucleic acid synthesis can be interrupted by severalechanisms
.3.1. FluoroquinolonesQuinolones or fluoroquinolones are among the most
mportant antibacterial drugs and are used extensively forhe treatment of bacterial infections both in human andeterinary medicine [86].
Fluoroquinolones exert their antibacterial effects bynhibition of certain bacterial topoisomerase enzymes,amely DNA gyrase and topoisomerase IV. DNA gyrase andopoisomerase IV are heterotetrameric proteins composed
f two subunits, designated A and B [87–90]. The genesncoding the A and B subunits are referred to as gyrA andyrB (DNA gyrase) or parC and parE (DNA topoisomeraseV), respectively (Table 2; Fig. 3d). DNA gyrase is the only
(crs
imicrobial Agents 32 (2008) 207–220 215
nzyme that can affect supercoiling of DNA, and inhibitionf this activity by fluoroquinolones is associated with rapidilling of the bacterial cell [2,91–93].
Alterations in target enzymes appear to be the most dom-nant factors in expression of resistance to quinolones [92].
small region from codon 67 to 106 of gyrA in E. coli wasesignated the ‘quinolone resistance-determining region’QRDR) [2] and variations in this region were found inpecies with natural resistance to fluoroquinolones [94,95].
Analysis of the T. whipplei genome allowed the identifi-ation of the gyrA and parC gene encoding the � subunit ofhe natural fluoroquinolone targets DNA gyrase and topoiso-
erase IV, respectively [16]. Heterogeneity in susceptibilityo fluoroquinolones in T. whipplei [16] was found to bessociated with mutations in the DNA gyrase gene. In the T.hipplei GyrA and ParC sequences, alanine residues were
ound at positions 81 and 96, respectively, correspondingo a serine at position 83 in E. coli GyrA [96] and a serinet position 80 in E. coli ParC [97], respectively (Table 3).yrA-mediated natural resistance to fluoroquinolones has
lso been described in Mycobacterium spp., which arelosely phylogenetically related to T. whipplei. In silicoenome analysis revealed a natural mutation at position 83f the QRDR region (Ser-83 → Ala) of the GyrA proteinor three Bartonella species (Table 3). Many examples existemonstrating that species naturally bearing a serine residuet position 83 of GyrA protein are usually susceptible touoroquinolones, whereas the presence of an alanine at thisritical position corresponds to natural resistance to thesentibiotics [98–101]. Similarly, Maurin et al. [102] observedhat a serine residue at position 83 of the GyrA protein inusceptible species of Ehrlichia is replaced by an alanineesidue in fluoroquinolone-resistant species.
.3.1.1. In vitro mutants. Resistance to fluoroquinolonesas been described in some strains of C. burnetii and it washown that the mechanism involved two distinct nucleotideutations in the GyrA protein (Glu-87 → Gly and Glu-
7 → Lys) [103,104] (Table 4). An amino acid changeAsp-87 → Asn) in its GyrA has also been reported recentlyn a ciprofloxacin-resistant strain of B. bacilliformis (Table 4).
.3.2. RifampicinThe molecular mechanism of rifampicin activity involves
nhibition of DNA-dependent RNA polymerase. This enzymes a complex oligomer composed of four different subunits (�,, �′ and � encoded by rpoA, rpoB, rpoC and rpoD, respec-
ively) (Fig. 3e). Rifampicin binds to the � subunit of RNAolymerase and results in transcription inhibition [105,106].
Resistance to rifampicin is primarily caused by mutationsn the rpoB gene. In the majority of rifampicin-resistantsolates, mutations occurred within an 81-bp hotspot region
the rifampicin resistance-determining region (RRDR),odons 507–533 according to E. coli numbering) in thepoB gene [4,107]. Previously, Rolain et al. [19] found thatusceptibility to rifampicin varied, with R. prowazekii, R.
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6. Efflux pumps
Efflux pumps are present in all living cells. They partici-pate in the detoxification process, expelling various harmful
16 S. Biswas et al. / International Journa
yphi, Rickettsia canada, Rickettsia bellii and most SFGickettsiae being susceptible to rifampicin, whilst the Rick-ttsia massiliae subgroup (R. massiliae, Rickettsia montana,ickettsia rhipicephalus and Rickettsia aeschlimannii)ere more resistant to rifampicin. Drancourt and Raoult
108] investigated the genetic basis for natural rifampicinesistance in representatives of the TG and the two SFGubgroups of rickettsiae by sequence analysis of the rpoBene. They found a single point mutation resulting in ahe → Leu change at position 973 of the R. conorii rpoBequence (Table 3). This single point mutation, whichppeared to be specific for the naturally rifampicin-resistantubgroup, was not previously implicated in rifampicinesistance in other bacteria. Resistance to rifampicin canlso be due to expression of an efflux system [109,110].
.3.2.1. In vitro mutants. Amino acid substitutions in theNA polymerase and rpoB point mutations have beenemonstrated following in vitro selection of rifampicin-esistant R. prowazekii [111] and R. typhi [112]. Troyer et al.112] reported the detection of a rifampicin-resistant strain of. typhi (Ethiopian). The basis of this resistance was inves-
igated by sequencing and mapping point mutations in thepoB gene of the mutant and then comparing the sequencesf wild-type and rifampicin-resistant R. typhi rpoB genes. Aotal of eight nucleotide substitutions occurred, three of whichesulted in amino acid substitutions in the mutant strain:eucine for phenylalanine at residue 151, phenylalanine foreucine at residue 201 and valine for isoleucine at residue71 [112] (Table 4). In another study by Rachek et al., com-arison of the rpoB sequences from the rifampicin-sensitive. prowazekii Madrid E strain and a rifampicin-resistantutant identified a single point mutation that resulted in an
rginine-to-lysine change at position 546 of the rpoB gene111].
A recently reported rifampicin-resistant strain of B.acilliformis from our group showed a mutation at serine31 (Ser → Phe) in the RRDR of the rpoB gene [53]Table 4). This 531 site is one of the most frequent sitesf mutation, also conferring rifampicin resistance in otheracterial species [106].
.4. Folate synthesis, which is necessary for DNAeplication, is blocked by sulphonamides andrimethoprim
.4.1. Trimethoprim and sulphonamidesTrimethoprim is an analogue of dihydrofolic acid, an
ssential component in the synthesis of amino acids anducleotides, which competitively inhibits the enzymeihydrofolate reductase (DHFR). Resistance can be caused
y a number of mechanisms, including overproduction ofost DHFR, mutations in the structural gene for DHFRnd acquisition of a foreign gene (dfr) encoding a resistantHFR enzyme [2,113,114].
Fo
imicrobial Agents 32 (2008) 207–220
The target for sulphonamide action is dihydropteroateynthase (DHPS), which catalyses the condensation ofara-aminobenzoic acid with 7,8-dihydro-6-hydroxy-ethylopterine pyrophosphate to form 7,8-dihydropteroate
115–118]. Sulphonamide resistance is commonly mediatedy the presence of alternative drug-resistant forms of DHPS.hromosomal mutations in the dhps gene that confer
esistance to sulphonamides have also been identified in aumber of bacteria [116,119].
Mutation in folA and folP (structural genes for DHFR andHPS, respectively) could confer resistance to trimethoprim
nd sulfamethoxazole, respectively. Interestingly, Rickettsiapp. are resistant to co-trimoxazole and it has been foundhat the folP and folA genes are absent in most Rickettsiapp. Coxiella burnetii, which is naturally susceptible too-trimoxazole compounds, showed both folP and folA genesn its genome. Interestingly, T. whipplei is susceptible too-trimoxazole, although only the folP gene is present in theropheryma genome. A recent study [120] demonstrated thathe MICs against the two strains of T. whipplei ranged from.5 mg/L to 1 mg/L for sulfadiazine compared with 0.5 mg/Lor sulfamethoxazole, leading the authors to suggest thatulfadiazine was as effective as sulfamethoxazole in vitro.
.4.1.1. In vitro study. We developed a new method to studyntibiotic susceptibility in fastidious bacteria such as T. whip-lei using E. coli gene complementation. In the genome of T.hipplei, a typical DHPS-encoding gene, the target gene for
ulfamethoxazole, is not found as an individual ORF. DNAequencing of two samples (before and after failure) from
patient with clinically acquired resistance to trimetho-rim/sulfamethoxazole, using specific oligonucleotiderimers for the candidate gene folP, showed three aminocid changes [121]. Gene complementation in E. coli showedhat the mutated sequence was associated with resistance.
ig. 5. Correlation between genome size of intracellular bacteria and numberf ATP-binding cassette (ABC) transporters present in the genome.
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S. Biswas et al. / International Journa
ompounds and xenobiotics [122,123]. Bacterial drug effluxumps are currently classified into five families [124–130]:i) the ATP-binding cassette (ABC) superfamily; (ii) MFSmajor facilitator superfamily) transporters; (iii) the RNDresistance–nodulation–cell division) superfamily; (iv) theMR (small multidrug resistance) family; and (v) the MATEmultidrug and toxic compound extrusion) family.
.1. In silico genome analysis
Whole genome analysis using an in silico method showedhat the number of genes encoding ABC transporters inntracellular bacteria varied from four (R. prowazekii, E.uminantium Welgevonden France and E. ruminantiumardel) to 277 (Brucella suis). We found a correlationetween the numbers of encoded ABC transporters andenome size, which is depicted in Fig. 5. Genome sizend number of ABC transporters in different intracellularacteria are given in Supplementary Data 1.
Several of the efflux systems found among intracellularacteria are given in Supplementary Data 2. Efflux systemsncluding pH adaptation potassium efflux system proteinsPhaAB, PhaC, PhaD, PhaE, PhaF and PhaG) have beenound in multiple different Bartonella spp. The genome of T.hipplei Twist contains the multidrug efflux protein QacAnd a cation efflux protein. The plasmid-encoded multidrugesistance gene, qacA, from Staphylococcus aureus mediatesesistance to a number of classes of antimicrobial organications, including intercalating dyes, quaternary ammoniumompounds, diamidines and biguanidines [70,76]. Interest-ngly, T. whipplei has been shown to be resistant to severalntiseptics, including glutaraldehyde and peracetic acid131].
In the genome of F. tularensis we found outer membranefflux proteins along with other efflux systems. The OEPouter membrane efflux protein) family forms trimerichannels allowing export of a variety of substrates inram-negative bacteria [132].The glutathione-regulated potassium efflux system
rotein, KefB, is present in the genome of all Rickettsia spp.nd is also known as K+/H+ antiporter. It is localised to thenner membrane of the cell and facilitates potassium efflux133].
. Concluding remarks and perspectives
The emergence and spread of antimicrobial resistanceeterminants continues to challenge our ability to treaterious infections. The past two decades have producedubstantial research into the mechanisms by which bacteriaevelop and disperse resistance determinants, although there
s still much to be learned. In silico genome analysis willelp to predict possible molecular mechanisms of resistancemong intracellular bacteria. Studying microbial genomesill also aid in the discovery process by identifying all bacte-
imicrobial Agents 32 (2008) 207–220 217
ial gene products that may be involved in antibiotic transportnd efflux from bacterial cells. Among intracellular bacteria,he spread of antibiotic resistance is mainly due to verticalene transfer, which is also observed in M. tuberculosis. Thisroup of organisms could be an interesting paradigm to iden-ify different resistance determinants using whole genomenalysis. Microarray studies could also be used to determinentibiotic resistance genes in intracellular bacteria. Thetudy of natural mutants and in vitro resistant mutants willdditionally help in the understanding of the efficacy ofifferent classes of antibiotics among intracellular bacteria.e developed a new method to study antibiotic resistance
n fastidious bacteria (e.g. T. whipplei) using E. coli geneomplementation. Transformation of intracellular bacterias one of the difficulties we face in working with theseicroorganisms. This complementation approach could be
sed with other intracellular bacteria for further study andould open a door to the identification and demonstrationf resistance determinants among these bacteria. Genomictudies will further clarify how resistance to novel classesf antibiotics arises, in addition to the fitness costs to therganism 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-le can be found, in the online version, atoi:10.1016/j.ijantimicag.2008.03.017.
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