International Journal of Antimicrobial Agents 32 (2008) 207220

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, Facult de Mdecine et de Pharmacie, Universit de la Mditerrane, 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 difcult 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 nd natural mutations in known target genes involved in antibiotic resistance and to look for the presence or absence of different 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 efux 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 Intracellular bacteria are dened 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 difcult 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: Unit des Rickettsies, CNRS UMR 6020, IFR 48, Facult de Mdecine et de Pharmacie, Universit de la Mditerrane, 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).

Treating bacterial infections is increasingly complicated 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 modication of the target of the antimicrobial agent, which reduces its binding capacity; (iv) reduced uptake of the antimicrobial agent; and (v) active efux 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

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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]. Resistanceassociated point mutations, deletions or insertions in M. tuberculosis have been previously described for all rst-line drugs (e.g. isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin) in addition to several second-line and newer drugs (e.g. ethionamide, uoroquinolones, 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.

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).

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 survival of intracellular bacteria: (a) survival in the cytoplasm
Table 1 Antibiotic susceptibility results for various intracellular bacteria Intracellular bacteria Antibiotic ERY Bartonella spp. Tropheryma whipplei Francisella tularensis Rickettsia spp. Typhus group R. prowazekii R. typhi Spotted fever group R. conorii subgroup R. conorii, R. rickettsii R. massiliae subgroup R. massiliae, R. montanensis Ehrlichia spp. E. canis E. chaffeensis Wolbachia spp. Coxiella burnetii Brucella spp. S S S/R AMG S S S TET S S S

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) survival in fused phagolysosomes (e.g. Coxiella, Tropheryma and Francisella) (Fig. 1). Antibiotic activity against intracellular bacteria depends on several factors, including pharmacodynamic and pharmacokinetic 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, uorometric and chemical

QUI S R S

CHL S S S

SXT S S R

RIF S S S

BL S S R

S S

R R

S S

S S

S S

R R

S S

R R

R R R R R S/R S/R

R R R R R R S

S S S S S S S

S S R R S/R S S

S S R R R R R

R R R R R S R

S R S S S S S

R R R R R R R

ERY, erythromycin; AMG, aminoglycosides; TET, tetracycline; QUI, quinolones; CHL, chloramphenicol; SXT, sulfamethoxazole/trimethoprim (cotrimoxazole); RIF, rifampicin; BL, -lactams; S, susceptible; R, resistant.

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techniques. For example, the uoroquinolones accumulate within phagocytes with a C/E ratio of 6:7 for granulocytes, 3:4 for macrophages and ca. 2:1 for epithelial cells [79]. 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. uoroquinolone 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

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, ow cytometry, immunouorescence techniques, 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 antibiotics among intracellular bacteria. Bacteria of the genus

Fig. 2. Phylogenetic tree based on 16S sequences of intracellular bacteria used in this study.

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S. Biswas et al. / International Journal of Antimicrobial Agents 32 (2008) 207220 Table 2 Candidate genes involved in antibiotic resistance Antibiotic Macrolides Tetracyclines Aminoglycosides Quinolones Rifampicin Chloramphenicol -Lactams Trimethoprim Sulfamethoxazole Candidate genes erm, mef, msr, 23S rRNA, rplD (L4 r-protein), rplV (L22 r-protein) 16S rRNA, tet gene, rpsL (S12 r-protein), rpsG (S7r-protein) 16S rRNA, rpsL (S12 r-protein), aminoglycoside-modifying enzymes gyrA, gyrB, parC, parE rpoB cat, 23S rRNA mecA folA folP

Bartonella are susceptible to all antibiotics in vitro, including -lactams, aminoglycosides, chloramphenicol, tetracyclines, macrolides, rifampicin, uoroquinolones and co-trimoxazole [13,14]. Tropheryma whipplei displays a homogeneous pattern of antibiotic susceptibility in axenic medium [15], with almost all antibiotics showing at least some activity, except uoroquinolones [16]. Francisella tularensis strains are susceptible to streptomycin, gentamicin, doxycycline, chloramphenicol and quinolones but show heterogeneous susceptibility 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 [2123] (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 bacteriostatic [24,25]. There is heterogeneity in susceptibility to erythromycin among the strains tested [25,26]. Strains of Brucella spp. also show heterogeneous susceptibility to erythromycin but are susceptible to almost all antibiotics except chloramphenicol, co-trimoxazole and -lactams [27,28].

erm, erythromycin ribosome methylation; mef, macrolide efux; msr, methionine sulfoxide reductase; r-protein, ribosomal protein; tet, tetracycline resistance protein; gyrA, DNA gyrase subunit A; gyrB, DNA gyrase subunit B; parC, DNA topoisomerase IV subunit A; parE, DNA topoisomerase 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. Macrolidelincosamidestreptogramin (MLS) antibiotics The MLS antibiotics are an important group of translation inhibitors that act on the 50S ribosomes [29]. The MLS group was dened 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 (macrolidelincosamide 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 [3032]. The rst described mechanism was a result of post-transcriptional modications of the 23S rRNA by adenine-N6 -methyltransferase. Ribosomal target modication 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 efux mediated by the membrane-bound efux protein encoded by mef(A), which confers resistance only to 14- and

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 available 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 possible mutations known to be associated with antibiotic resistance. The keywords used for the in silico genome study were: efux; 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; uoroquinolone; 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

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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 uoroquinolones 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-specic efux proteins (e.g. MacA and MacB) were identied in the Bartonella genome. In the Escherichia coli genome, ybjYZ were suspected to be genes for ABC drug efux transporters and were renamed macAB (macrolide-specic ABC-type efux 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

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5990 and 8587 in the L4 and L22 ribosomal proteins, respectively, have been reported to be important mutational regions for macrolide resistance [39,4143]. 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] conrmed that macrolides and telithromycin lacked antimicrobial activity against Ehrlichia [2123,45]. They reported numerous specic mutations in nucleotides known to confer resistance to macrolides in the Ehrlichia chaffeensis 23S rRNA gene (e.g. T754G, G2057A, A2059G and C2611T) [44]. Wolbachia 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 inuenzae 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., different 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 erythromycin resistance in other bacteria as well [34,39,44,52]. We found amino acid mutations at two different positions (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; mutation 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 chloramphenicol 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 modication by specic enzymes produced by resistant organisms. The three classes of such aminoglycoside-modifying enzymes are: (1) aminoglycoside nucleotidyltransferases, which transfer nucleotide triphosphates; (2) aminoglycoside acetyltransferases, which transfer the acetyl group from acetyl-CoA; and (3) aminoglycoside phosphotransferases, which transfer the phosphoryl group from ATP [5861]. 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

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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 efux pump (AcrD, acriavine 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 aminoglycosidebinding site, and reduction of the intracellular concentration of aminoglycosides by changes in outer membrane permeability, decreased inner membrane transport and active efux [6369]. 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, highafnity 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 rst group of genes encodes efux systems that transport the drug from the inside to the outside of the bacterial cell; the second

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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 23S rRNA Bartonella henselae Bartonella quintana Bartonella bacilliformis Rickettsia spp. Francisella tularensis Ehrlichia chaffeensis A2059G L22 ribosomal protein FQa gyrA Ser-83 Ala Ser-83 Ala Ser-83 Ala Triple AA differences A2059C T754G G2057A A2059G C2611T T754A A2058G A2059C C2611G Ser-83 Ala APH parC AMG BL RIF (rpoB)a CHL

MBL BLA BLA MBL

Phe-973 Leu G2057A

Wolbachia pipientis

MBL

Tropheryma whipplei Coxiella burnetii Brucella suis

Ser-83 Ala

Ser-96 Ala AAC MBL MBL

MAC, macrolides; FQ, uoroquinolones; AMG, aminoglycosides; BL, -lactams; RIF, rifampicin; CHL, chloramphenicol; AA, amino acid; APH, aminoglycoside 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 efux 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 tetracycline 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; -lactam drugs such as penicillins, cephalosporins and carbapenems all interfere with cell wall production 5.2.1. -Lactam antibiotics -Lactam antibiotics are among the most commonly used antimicrobial agents. They interfere with the nal 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 23S rRNA Bartonella henselae A2058G A2058C C2611T A2058G L4 ribosomal protein G71R H75Y Insertion GRARHSSAR Asp-87 Asn Ser-531 Phe Leu-151 Phe Phe-201 Leu Val-271 Ile Arg-546 Lys Val-57 Ile Thr-102 Pro Leu-162 Ile FQ (gyrA)a RIF (rpoB)a SMX (dhps)a

Bartonella quintana Bartonella bacilliformis Rickettsia spp.

Francisella tularensis Coxiella burnetii Tropheryma whipplei

Thr-83 Ile Glu-87 Gly

MAC, macrolides; FQ, uoroquinolones; RIF, rifampicin; SMX, sulfamethoxazole. a Amino acid changes resulting from the gene mutation are given.

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of cell wall synthesis by inhibiting the bacterial enzymes transpeptidases and carboxypeptidases that catalyse the reactions 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 antibiotics is antibiotic-inactivating enzymes, the -lactamases, which efciently 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 proles, inhibition proles, molecular mass, isoelectric point, amino acid sequence and molecular structure [77,81,82]. Genes encoding -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 Ambers 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-specic 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 specic open-reading frames (ORFs) related to antibiotic resistance have been previously identied 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 uoroquinolones 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 [8790]. 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

enzyme that can affect supercoiling of DNA, and inhibition of this activity by uoroquinolones is associated with rapid killing of the bacterial cell [2,9193]. Alterations in target enzymes appear to be the most dominant 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 uoroquinolones [94,95]. Analysis of the T. whipplei genome allowed the identication of the gyrA and parC gene encoding the subunit of the natural uoroquinolone targets DNA gyrase and topoisomerase IV, respectively [16]. Heterogeneity in susceptibility to uoroquinolones 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 uoroquinolones 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 uoroquinolones, whereas the presence of an alanine at this critical position corresponds to natural resistance to these antibiotics [98101]. 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 uoroquinolone-resistant species. 5.3.1.1. In vitro mutants. Resistance to uoroquinolones 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 Glu87 Lys) [103,104] (Table 4). An amino acid change (Asp-87 Asn) in its GyrA has also been reported recently in a ciprooxacin-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, respectively) (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 507533 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.

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typhi, Rickettsia canada, Rickettsia bellii and most SFG rickettsiae being susceptible to rifampicin, whilst the Rickettsia massiliae subgroup (R. massiliae, Rickettsia montana, Rickettsia rhipicephalus and Rickettsia aeschlimannii) were more resistant to rifampicin. Drancourt and Raoult [108] investigated the genetic basis for natural rifampicin resistance in representatives of the TG and the two SFG subgroups of rickettsiae by sequence analysis of the rpoB gene. They found a single point mutation resulting in a Phe Leu change at position 973 of the R. conorii rpoB sequence (Table 3). This single point mutation, which appeared to be specic for the naturally rifampicin-resistant subgroup, was not previously implicated in rifampicin resistance in other bacteria. Resistance to rifampicin can also be due to expression of an efux system [109,110]. 5.3.2.1. In vitro mutants. Amino acid substitutions in the RNA polymerase and rpoB point mutations have been demonstrated following in vitro selection of rifampicinresistant R. prowazekii [111] and R. typhi [112]. Troyer et al. [112] reported the detection of a rifampicin-resistant strain of R. typhi (Ethiopian). The basis of this resistance was investigated by sequencing and mapping point mutations in the rpoB gene of the mutant and then comparing the sequences of wild-type and rifampicin-resistant R. typhi rpoB genes. A total of eight nucleotide substitutions occurred, three of which resulted in amino acid substitutions in the mutant strain: leucine for phenylalanine at residue 151, phenylalanine for leucine at residue 201 and valine for isoleucine at residue 271 [112] (Table 4). In another study by Rachek et al., comparison of the rpoB sequences from the rifampicin-sensitive R. prowazekii Madrid E strain and a rifampicin-resistant mutant identied a single point mutation that resulted in an arginine-to-lysine change at position 546 of the rpoB gene [111]. A recently reported rifampicin-resistant strain of B. bacilliformis from our group showed a mutation at serine 531 (Ser Phe) in the RRDR of the rpoB gene [53] (Table 4). This 531 site is one of the most frequent sites of mutation, also conferring rifampicin resistance in other 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 DHFR enzyme [2,113,114].

The target for sulphonamide action is dihydropteroate synthase (DHPS), which catalyses the condensation of para-aminobenzoic acid with 7,8-dihydro-6-hydroxymethylopterine pyrophosphate to form 7,8-dihydropteroate [115118]. Sulphonamide resistance is commonly mediated by the presence of alternative drug-resistant forms of DHPS. Chromosomal mutations in the dhps gene that confer resistance to sulphonamides have also been identied in a number of bacteria [116,119]. Mutation in folA and folP (structural genes for DHFR and DHPS, respectively) could confer resistance to trimethoprim and sulfamethoxazole, respectively. Interestingly, Rickettsia spp. are resistant to co-trimoxazole and it has been found that the folP and folA genes are absent in most Rickettsia 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 co-trimoxazole, although only the folP gene is present in the Tropheryma genome. A recent study [120] demonstrated that the MICs against the two strains of T. whipplei ranged from 0.5 mg/L to 1 mg/L for sulfadiazine compared with 0.5 mg/L for sulfamethoxazole, leading the authors to suggest that sulfadiazine was as effective as sulfamethoxazole in vitro. 5.4.1.1. In vitro study. We developed a new method to study antibiotic susceptibility in fastidious bacteria such as T. whipplei using E. coli gene complementation. In the genome of T. whipplei, a typical DHPS-encoding gene, the target gene for sulfamethoxazole, is not found as an individual ORF. DNA sequencing of two samples (before and after failure) from a patient with clinically acquired resistance to trimethoprim/sulfamethoxazole, using specic oligonucleotide primers for the candidate gene folP, showed three amino acid changes [121]. Gene complementation in E. coli showed that the mutated sequence was associated with resistance.

6. Efux pumps Efux pumps are present in all living cells. They participate in the detoxication process, expelling various harmful

Fig. 5. Correlation between genome size of intracellular bacteria and number of ATP-binding cassette (ABC) transporters present in the genome.

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compounds and xenobiotics [122,123]. Bacterial drug efux pumps are currently classied into ve families [124130]: (i) the ATP-binding cassette (ABC) superfamily; (ii) MFS (major facilitator superfamily) transporters; (iii) the RND (resistancenodulationcell 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 efux systems found among intracellular bacteria are given in Supplementary Data 2. Efux systems including pH adaptation potassium efux 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 efux protein QacA and a cation efux 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]. Interestingly, 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 efux proteins along with other efux systems. The OEP (outer membrane efux protein) family forms trimeric channels allowing export of a variety of substrates in Gram-negative bacteria [132]. The glutathione-regulated potassium efux 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 efux [133].

rial gene products that may be involved in antibiotic transport and efux 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 identify 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 efcacy 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 difculties 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 identication 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 tness 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 article can be found, in the online version, at doi:10.1016/j.ijantimicag.2008.03.017.

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