Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing. - Related Documents




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482801.0000Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing. Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The "last-line" antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.202032716393
438310.9999Importance of Core Genome Functions for an Extreme Antibiotic Resistance Trait. Extreme antibiotic resistance in bacteria is associated with the expression of powerful inactivating enzymes and other functions encoded in accessory genomic elements. The contribution of core genome processes to high-level resistance in such bacteria has been unclear. In the work reported here, we evaluated the relative importance of core and accessory functions for high-level resistance to the aminoglycoside tobramycin in the nosocomial pathogen Acinetobacter baumannii Three lines of evidence establish the primacy of core functions in this resistance. First, in a genome scale mutant analysis using transposon sequencing and validation with 594 individual mutants, nearly all mutations reducing tobramycin resistance inactivated core genes, some with stronger phenotypes than those caused by the elimination of aminoglycoside-inactivating enzymes. Second, the core functions mediating resistance were nearly identical in the wild type and a deletion mutant lacking a genome resistance island that encodes the inactivating enzymes. Thus, most or all of the core resistance determinants important in the absence of the enzymes are also important in their presence. Third, reductions in tobramycin resistance caused by different core mutations were additive, and highly sensitive double and triple mutants (with 250-fold reductions in the MIC) that retained accessory resistance genes could be constructed. Core processes that contribute most strongly to intrinsic tobramycin resistance include phospholipid biosynthesis, phosphate regulation, and envelope homeostasis.IMPORTANCE The inexorable increase in bacterial antibiotic resistance threatens to undermine many of the procedures that transformed medicine in the last century. One strategy to meet the challenge antibiotic resistance poses is the development of drugs that undermine resistance. To identify potential targets for such adjuvants, we identified the functions underlying resistance to an important class of antibiotics for one of the most highly resistant pathogens known.201729233894
438120.9998Specific Gene Loci of Clinical Pseudomonas putida Isolates. Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.201626820467
482730.9998A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii. Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.201526170289
989740.9998The fitness connection of antibiotic resistance. More than three decades ago multidrug-resistant (MDR) clones of the pathogens: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Clostridioides difficile, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii have started to disseminate across wide geographical areas. A characteristic feature of all these MDR lineages is the carriage of some mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV which besides conferring resistance to fluoroquinolones are associated with a fitness benefit. Several lines of evidence strongly suggest that extra fitness conferred by these mutations facilitated the dissemination of the international MDR lineages. MDR pathogens require extra energy to cover the fitness cost conferred by the excess antibiotic resistance gene cargo. However, extra energy generated by upgraded metabolic activity was demonstrated to increase the uptake of antibiotics enhancing susceptibility. Accordingly, MDR bacteria need additional positive fitness schemes which, similarly to the QRDR advantage, will not compromise resistance. Some of these, not clone-specific effects are large genomes, the carriage of low-cost plasmids, the transfer of plasmid genes to the chromosome, the application of weak promoters in integrons and various techniques for the economic control of the activity of the integrase enzyme including a highly sophisticated system in A. baumannii. These impacts - among others - will confer a fitness advantage promoting the spread of MDR pathogens. However, even the potential of extra fitness generated by the combined effect of various schemes is not without limit and virulence-related genes or less relevant antibiotic resistance gene cargoes will often be sacrificed to permit the acquisition of high-priority resistance determinants. Accordingly major MDR clone strains are usually less virulent than susceptible isolates. In summary, a fitness approach to the research of antibiotic resistance is very useful since the fitness status of MDR bacteria seem to profoundly impact the capacity to disseminate in the healthcare setting.202540276228
442950.9998General mechanisms of resistance to antibiotics. Resistance to antimicrobial agents may result from intrinsic properties of organisms, through mutation and through plasmid- and transposon-specified genes. beta-Lactam resistance is most frequently associated with one or more chromosomal- or plasmid-specified beta-lactamases. Recently, mutations modifying penicillin-binding proteins have been detected with increased frequency as a cause of beta-lactam resistance. Mixed mechanisms, reduced permeability and tolerance are other causes of resistance. Aminoglycoside resistance always involves some modification of drug uptake, most often due to a variety of enzymes modifying these compounds. Reduced uptake is a primary cause of resistance in anaerobic bacteria and bacteria growing anaerobically, some strains of Pseudomonas aeruginosa, and mutants that arise during antimicrobial therapy and are defective in energy-generation systems. Resistance to other antimicrobial agents is presented in tabular form.19883062000
940460.9998The Application of Transposon Insertion Sequencing in Identifying Essential Genes in B. fragilis. Essential genes are those that are indispensable for the survival of organism under specific growth conditions. Investigating essential genes in pathogenic bacteria not only helps to understand vital biological networks but also provides novel targets for drug development. Availability of genetic engineering tools and high-throughput sequencing methods has enabled essential genes identification in many pathogenic gram-positive and gram-negative bacteria. Bacteroides fragilis is one of the major bacteria specific of human gastrointestinal microbiota. When B. fragilis moves out of its niche, it turns into deadly pathogen. Here, we describe detailed method for the essential gene identification in B. fragilis. Generated transposon mutant pool can be used for other applications such as identification of genes responsible for drug resistance in B. fragilis.202234709623
633570.9998Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli. The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.202134756069
426380.9998The emergence of antibiotic resistance by mutation. The emergence of mutations in nucleic acids is one of the major factors underlying evolution, providing the working material for natural selection. Most bacteria are haploid for the vast majority of their genes and, coupled with typically short generation times, this allows mutations to emerge and accumulate rapidly, and to effect significant phenotypic changes in what is perceived to be real-time. Not least among these phenotypic changes are those associated with antibiotic resistance. Mechanisms of horizontal gene spread among bacterial strains or species are often considered to be the main mediators of antibiotic resistance. However, mutational resistance has been invaluable in studies of bacterial genetics, and also has primary clinical importance in certain bacterial species, such as Mycobacterium tuberculosis and Helicobacter pylori, or when considering resistance to particular antibiotics, especially to synthetic agents such as fluoroquinolones and oxazolidinones. In addition, mutation is essential for the continued evolution of acquired resistance genes and has, e.g., given rise to over 100 variants of the TEM family of beta-lactamases. Hypermutator strains of bacteria, which have mutations in genes affecting DNA repair and replication fidelity, have elevated mutation rates. Mutational resistance emerges de novo more readily in these hypermutable strains, and they also provide a suitable host background for the evolution of acquired resistance genes in vitro. In the clinical setting, hypermutator strains of Pseudomonas aeruginosa have been isolated from the lungs of cystic fibrosis patients, but a more general role for hypermutators in the emergence of clinically relevant antibiotic resistance in a wider variety of bacterial pathogens has not yet been proven.200717184282
630890.9998A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa. BACKGROUND: Antibiotics in current use target a surprisingly small number of cellular functions: cell wall, DNA, RNA, and protein biosynthesis. Targeting of novel essential pathways is expected to play an important role in the discovery of new antibacterial agents against bacterial pathogens, such as Pseudomonas aeruginosa, that are difficult to control because of their ability to develop resistance, often multiple, to all current classes of clinical antibiotics. RESULTS: We aimed to identify novel essential genes in P. aeruginosa by shotgun antisense screening. This technique was developed in Staphylococcus aureus and, following a period of limited success in Gram-negative bacteria, has recently been used effectively in Escherichia coli. To also target low expressed essential genes, we included some variant steps that were expected to overcome the non-stringent regulation of the promoter carried by the expression vector used for the shotgun antisense libraries. Our antisense screenings identified 33 growth-impairing single-locus genomic inserts that allowed us to generate a list of 28 "essential-for-growth" genes: five were "classical" essential genes involved in DNA replication, transcription, translation, and cell division; seven were already reported as essential in other bacteria; and 16 were "novel" essential genes with no homologs reported to have an essential role in other bacterial species. Interestingly, the essential genes in our panel were suggested to take part in a broader range of cellular functions than those currently targeted by extant antibiotics, namely protein secretion, biosynthesis of cofactors, prosthetic groups and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcription regulation. This study also identified 43 growth-impairing inserts carrying multiple loci targeting 105 genes, of which 25 have homologs reported as essential in other bacteria. Finally, four multigenic growth-impairing inserts belonged to operons that have never been reported to play an essential role. CONCLUSIONS: For the first time in P. aeruginosa, we applied regulated antisense RNA expression and showed the feasibility of this technology for the identification of novel essential genes.201424499134
4428100.9998Multidrug resistance in enteric and other gram-negative bacteria. In Gram-negative bacteria, multidrug resistance is a term that is used to describe mechanisms of resistance by chromosomal genes that are activated by induction or mutation caused by the stress of exposure to antibiotics in natural and clinical environments. Unlike plasmid-borne resistance genes, there is no alteration or degradation of drugs or need for genetic transfer. Exposure to a single drug leads to cross-resistance to many other structurally and functionally unrelated drugs. The only mechanism identified for multidrug resistance in bacteria is drug efflux by membrane transporters, even though many of these transporters remain to be identified. The enteric bacteria exhibit mostly complex multidrug resistance systems which are often regulated by operons or regulons. The purpose of this review is to survey molecular mechanisms of multidrug resistance in enteric and other Gram-negative bacteria, and to speculate on the origins and natural physiological functions of the genes involved.19968647368
4405110.9998Copper Resistance of the Emerging Pathogen Acinetobacter baumannii. Acinetobacter baumannii is an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. Since A. baumannii can rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies against Acinetobacter infections, we characterized copper resistance in a panel of recent clinical A. baumannii isolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper on A. baumannii revealed that resistance to copper increased dramatically in stationary phase. Moreover, A. baumannii biofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins in Escherichia coli and Pseudomonas aeruginosa Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics against Acinetobacter and other highly antibiotic-resistant pathogens. IMPORTANCE: Acinetobacter baumannii causes many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options are incredibly limited. Copper is an essential nutrient but becomes toxic at high concentrations. The inherent antimicrobial properties of copper give it potential for use in novel therapeutics against drug-resistant pathogens. We show that A. baumannii clinical isolates are sensitive to copper in vitro, both in liquid and on solid metal surfaces. Since bacterial resistance to copper is mediated though mechanisms of efflux and detoxification, we identified genes encoding putative copper-related proteins in A. baumannii and showed that expression of some of these genes is regulated by the copper concentration. We propose that the antimicrobial effects of copper may be beneficial in the development of future therapeutics that target multidrug-resistant bacteria.201627520808
4832120.9998Antibiotic resistance of Pseudomonas species. Pseudomonas species are highly versatile organisms with genetic and physiologic capabilities that allow them to flourish in environments hostile to most pathogenic bacteria. Within the lung of the patient with cystic fibrosis, exposed to a number of antimicrobial agents, highly resistant clones of Pseudomonas are selected. These may have acquired plasmid-mediated genes encoding a variety of beta-lactamases or aminoglycoside modifying enzymes. Frequently these resistance determinants are on transposable elements, facilitating their dissemination among the population of bacteria. Mutations in chromosomal genes can also occur, resulting in constitutive expression of normally repressed enzymes, such as the chromosomal cephalosporinase of Pseudomonas aeruginosa or Pseudomonas cepacia. These enzymes may confer resistance to the expanded-spectrum beta-lactam drugs. Decreased cellular permeability to the beta-lactams and the aminoglycosides also results in clinically significant antibiotic resistance. The development of new drugs with anti-Pseudomonas activity, beta-lactam agents and the quinolones, has improved the potential for effective chemotherapy but has not surpassed the potential of the organisms to develop resistance.19863701534
3808130.9998Expression Profiling of Antibiotic-Resistant Bacteria Obtained by Laboratory Evolution. To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and genotypic features in resistant strains is important. Here, we describe the expression profiling of antibiotic-resistant Escherichia coli strains obtained by laboratory evolution, and a method for extracting a small number of genes whose expression changes can contribute to the acquisition of resistance.201727873258
9405140.9998Functional Metagenomic Screening for Antimicrobial Resistance in the Oral Microbiome. A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance.202134410638
4249150.9998Detection of essential genes in Streptococcus pneumoniae using bioinformatics and allelic replacement mutagenesis. Although the emergence and spread of antimicrobial resistance in major bacterial pathogens for the past decades poses a growing challenge to public health, discovery of novel antimicrobial agents from natural products or modification of existing antibiotics cannot circumvent the problem of antimicrobial resistance. The recent development of bacterial genomics and the availability of genome sequences allow the identification of potentially novel antimicrobial agents. The cellular targets of new antimicrobial agents must be essential for the growth, replication, or survival of the bacterium. Conserved genes among different bacterial genomes often turn out to be essential (1, 2). Thus, the combination of comparative genomics and the gene knock-out procedure can provide effective ways to identify the essential genes of bacterial pathogens (3). Identification of essential genes in bacteria may be utilized for the development of new antimicrobial agents because common essential genes in diverse pathogens could constitute novel targets for broad-spectrum antimicrobial agents.200818392984
8915160.9998Genetic regulation of host responses to Salmonella infection in mice. Salmonella spp are Gram-negative bacteria capable of infecting a wide range of host species, including humans, domesticated and wild mammals, reptiles, birds and insects. The outcome of an encounter between Salmonella and its host is dependent upon multiple factors including the host genetic background. To facilitate the study of the genetic factors involved in resistance to this pathogen, mouse models of Salmonella infection have been developed and studied for years, allowing identification of several genes and pathways that may influence the disease outcome. In this review, we will cover some of the genes involved in mouse resistance to Salmonella that were identified through the study of congenic mouse strains, cloning of spontaneous mouse mutations, use of site-directed mutagenesis or quantitative trait loci analysis. In parallel, the relevant information pertaining to genes involved in resistance to Salmonella in humans will be discussed.200212424619
3824170.9998Screening for novel antibiotic resistance genes. Knowledge of novel antibiotic resistance genes aids in the understanding of how antibiotics function and how bacteria fight them. This knowledge also allows future generations of an antibiotic or antibiotic group to be altered to allow the greatest efficacy. The method described here is very simple in theory. The bacterial strains are screened for antibiotic resistance. Cultures of the strain are grown, and DNA is extracted. A partial digest of the extraction is cloned into Escherichia coli, and the transformants are plated on selective media. Any colony that grows will possess the antibiotic resistance gene and can be further examined. In actual practice, however, this technique can be complicated. The detailed protocol will need to be optimized for each bacterial strain, vector, and cell line chosen.201020830570
9310180.9998Bacterial resistance to antibiotics. Effective antibacterial drugs have been available for nearly 50 years. After the introduction of each new such drug, whether chemically synthesized or a naturally occurring antibiotic, bacterial resistance to it has emerged. The genetic mechanisms by which bacteria have acquired resistance were quite unexpected; a new evolutionary pathways has been revealed. Although some antibiotic resistance has resulted from mutational changes in structural proteins--targets for the drugs' action--most has resulted from the acquisition of new, ready-made genes from an external source--that is, from another bacterium. Vectors of the resistance genes are plasmids--heritable DNA molecules that are transmissible between bacterial cells. Plasmids without antibiotic-resistance genes are common in all kinds of bacteria. Resistance plasmids have resulted from the insertion of new DNA sequences into previously existing plasmids. Thus, the spread of antibiotic resistance is at three levels: bacteria between people or animals; plasmids between bacteria; and transposable genes between plasmids.19846319093
4382190.9998A bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis. 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 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-beta-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.200818619818