Rustam I. Aminov, Joanne C. Chee-Sanford, Natalie Garrigues, Asma Mehboob, and Roderick I. Mackie
Rapid, accurate, and sensitive determination of antibiotic resistance profiles of various human and animal pathogens becomes a vital prerequisite for successful therapeutic intervention in the face of the increased occurrences of drug-resistant bacterial infections. The current methods, which are dependent on cultivation of pathogens and phenotypic expression of antibiotic resistance, usually require excessive time, special microbiological equipment, and qualified personnel. However, even with all these requisites, for example, no bacteria can be grown from more than 80% of all clinical samples sent to clinical microbiology laboratories (1). Besides the cultivation limitations, the cultivation-based determination of an antibiotic resistance profile lacks the genotypic information, which is essential for understanding the epidemiology and routes of transmission of antibiotic resistance genes. These genes often reside on mobile genetic elements and can move freely between commensal and pathogenic microbiota, occurring even between taxonomically distant clinical and environmental microbiota. Therefore, development of genotyping methods for detection of antibiotic resistance genes is highly desirable for fast, accurate, and sensitive detection of antibiotic resistance genes in a broad range of pathogenic and commensal bacteria in both clinical and environmental samples.
As a model for our studies we have chosen the genes conferring resistance to tetra-cyclines. Tetracyclines belong to a family of broad-spectrum antibiotics that include tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxy-cycline, minocycline, and a number of other semisynthetic derivatives (2). These antibiotics inhibit protein synthesis in Gram-positive and Gram-negative bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit (3). The antibiotics of this group were introduced in the late 1950s and since then have been widely used in clinical and veterinary medicine, as well as for prophylaxis and
From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ
growth promotion in food animals. Because of the possible misuse and overuse of these drugs, resistance to this class of antibiotics is widespread among many clinical isolates, thus limiting the utility of tetracyclines in treating infections. Despite this shortcoming, antibiotics of this class still remain in the active arsenal for dermatologists to treat skin infections such as acne (4) and rosacea (5). The local application of high concentrations of doxycycline has been proposed for clinical management of patients with periodontitis (6). Currently, the third generation of tetracyclines, the so-called glycylcyclines, are in phase II clinical trials (2), but microbial resistance to these novel antibiotics can already be seen among Salmonella isolates (7). Interestingly, this resistance to glycylcyclines is mediated by the well-known tetracycline resistance gene, tet(A), which has acquired an additional mutation conferring resistance to novel antibiotics that have not yet been introduced into clinical practice. Thus, routine monitoring for existing and novel tetracycline resistance genes may aid in evaluating possible decreases in efficacy of third-generation tetracyclines caused by spreading resistances.
Bacterial resistance to tetracycline is mediated mainly by two mechanisms including protection of ribosomes by the synthesis of ribosomal protection proteins (RPPs), which share homology with the GTPases involved in protein synthesis, namely, EF-Tu and EF-G (8-14), and by the energy-dependent efflux of tetracycline from the cell (3,12,15). A third mechanism, enzymatic inactivation of tetracycline, is relatively uncommon and functions only during heterologous expression in E. coli (16). This reaction requires the presence of oxygen and NADPH and is not functional in the natural anaerobic host (Bacteroides). The first nomenclature for tetracycline resistance determinants was proposed in 1989 (17), and a recent update appeared in 1999 (18). Phylogeny-based classifications of the RPP genes allowed the identification of nine classes: TetB P, TetM, TetO, TetQ, TetS, TetT, TetW, Tet 32, and otrA (19,20). The second mechanism of tetracycline resistance, efflux of tetracycline from the cell, is mediated by transporters, which share a common structure with the 12 or 14 transmembrane segments (12-TMS and 14-TMS) and belong to the major facilitator super-family (MFS) (21,22). The 12-TMS permeases are found almost exclusively in Gram-negative bacteria and they uniformly catalyze drug/H+ antiport (21). Recent phylogenetic analysis has suggested that the tet subcluster includes 11 genes catalyzing the efflux of tetracycline from the cell: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30) (23).
Genotyping of antibiotic resistance genes can be done by hybridization with any of the known tet genes or by polymerase chain reaction (PCR). A hybridization approach is time-consuming and is less sensitive when one is working with environmental or clinical samples. Based on the phylogenetic analysis of tetracycline resistance genes, we have developed PCR primer sets suitable for detection and tracking of these genes in various bacteria and environmental samples (19,23,24) as well as for quantification of these genes in a variety of bacterial isolates and samples (unpublished data). This PCR-based approach can be easily automated and scaled up, and a similar detection approach can be adapted for detection of other, more clinically relevant, antibiotic resistance genes.
1. Depending on the type of samples used for PCR analysis, genomic DNA can be isolated using the kits from Mo Bio Laboratories (Solana Beach, CA, www.mobio.com):
a. From tissues, bones, and cell cultures: UltraClean™ Tissue DNA Kit.
b. From whole blood: UltraClean BloodSpin Kit or UltraClean™ Blood DNA Kit.
c. From plant tissue: UltraClean Plant DNA Kit.
d. From soil samples: UltraClean Soil DNA Kit.
e. From water and urine samples: UltraClean Water DNA Kit.
f. From fecal samples: UltraClean DRY Soil DNA Kit for fecal samples.
g. From microorganisms: UltraClean Microbial DNA Kit.
h. From forensic samples: UltraClean Forensic DNA Kit.
Additional equipment for genomic DNA isolation using these kits includes a vortex with a flat-bed pan for processing of multiple samples and a microcentrifuge.
2. PCR primers for detection of the RPP genes (Table 1) or tetracycline efflux pump genes of Gram-negative bacteria (TEPGNB) (Table 2) are synthesized commercially on a 50-nmol scale (high performance liquid chromatography [HPLC] purified). For PCR-denaturing gradient gel electrophoresis (DGGE) analysis, the forward or reverse primer is synthesized with a GC clamp (CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGC ACGGGGGG) attached to the 5'-end.
3. Takara Ex Taq™ DNA polymerase kit (Takara Shuzo, Japan).
4. Agarose gel electrophoresis buffer: 45 mM tris-borate, 1 mM EDTA, pH 8.0. This buffer should not require pH adjustment. The 10X stock is stored at room temperature and is used at a working strength of 0.5X.
5. Agarose gel sample loading buffer (6X): 40% (w/v) sucrose in water and 0.5% (w/v) Orange G (Sigma). This dye migrates as a DNA fragment of approx 50 bp.
6. Agarose gel stain solution: 0.5 mg/L (w/v) ethidium bromide in water. Alternatively, for faster results, the fluorescent dye GelStar® (FMC Bioproducts, Rockland, ME) can be incorporated into the agarose gels at a 1:10,000 dilution (v/v).
7. 100-bp DNA Ladder (Promega).
8. DGGE buffer: 40 mM Tris-acetate, 1 mM EDTA, pH 8.0. The 50X stock solution is stored at room temperature and is used at a working strength of 1X.
9. DGGE sample loading buffer: 0.05% bromophenol blue, 0.05% xylene cyanol, and 70% glycerol in sterile nanopure water.
10. GelBond PAG gel support films (FMC Bioproducts).
11. Freshly prepared silver stain solution: 0.1% AgNO3 in ddH2O.
12. Developing solution: 0.01% NaBH4 and 0.4% v/v formaldehyde in 1.5% w/v NaOH.
13. QuantiTect SYBR Green PCR kit (Qiagen, Germany) or other high-sensitivity real-time PCR kits based on quantification of amplified DNA by binding the SYBR Green fluorescent dye.
14. Control strains to generate the standard curves for quantification of tetracycline resistance genes in the environmental samples or for determining the copy number of tetracy-cline resistance genes in microbial cells (Table 3). These strains are also used as controls in PCR and PCR-DGGE.
15 QIAquick Gel Extraction Kit (Qiagen).
PCR Primers Targeting the Ribosomal Protection Protein Genes
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