Resistance to glycopeptides (vancomycin and teicoplanin) in Enterococcus spp. Phenotypes VanA, VanB, vand, and other less frequent (VanC, C2 / 3, E, F, G, L, M) - Molecular diagnosis (PCR and sequencing).
Glycopeptide resistance in Enterococcus spp. It is a major problem since its inception in the 1980s because of its importance as a major nosocomial pathogen. Glycopeptides vancomycin and teicoplanin, act by binding to the terminal dimer D-alanyl-D-alanine (D-Ala-D-Ala) of the final precursor for the formation of the peptidoglycan by inhibiting the formation of cross - links, by blocking the transglucosylation and transpeptidation.
Resistance in Enterococcus spp. It is a result of the synthesis of final precursors of peptidoglycan with low affinity to these antibiotics because these bacteria can synthesize a final precursor in which the native dimer D-Ala-D-Ala is replaced by other dimers as D-Ala- D-Lac (D-alanyl-D-lactose) or D-Ala-D-Ser (D-alanyl-D-serine). This new synthesis is mediated by group (cluster) of genes called van. They described seven kinds of sets of genes conferring resistance to Enterococcus where there are common genes, but also different genes and even different organization within the gene set. The types of gene sets (cluster) (resistance operon glycopeptides) are named according to the name of the gene encoding the ligase. There are up to 8 different types of ligase, although they differ in their sequences, can differentiate into the leading end dimer precursor: ligases D-Ala: D-Lac that generate the dimer D-Ala-D-Lac (encoded by vanA genes, vanB and vand), or D-Ala ligases: D-Ser, which leads to the final dimer precursor D-Ala-D-Ser (encoded by genes vanC, vanE, vanG and vanL).
Genes in different types "are"
Gene sets of vanA types, vanB and vand, generators dimer D-Ala-D-Lac contain genes for:
- Two-component regulatory system: VanR (response regulator) and vans. (histidinaquinasa sensor). These genes controlling expression of resistance operon.
- Resistance genes: vanH (encoding a dehydrogenase that reduces pyruvate to lactate, may be called vanH, vanH B, vanH D); vanA, vanB or vand ligase encoding type D-Ala: D-Lac.
- Accessory genes: vanX (D, D-dipeptidase, whose function is to hydrolyze the dimer D-Ala-D-Ala precursor, may be called vanX, vanX B, vanX D); VaNy (D, D-carboxypeptidase, whose function is to cleave the D-alanine last residue C-terminal dimer D-Ala-D-Ala; may be called VaNy, VaNy B, VaNy D); Vanz gene (present in whole vanA type); vanW gene (present in whole vanB type).
- Gene of unknown function: vanW (in one set of vanB type) .; Vanz (only in the set of VanA type).
Gene sets types vanC, vanE, vanG and vanL generators dimer D-Ala-D-Ser, containing genes for:
- Two-component regulatory system: VanR (response regulator, such VanR C-2) and vans (histidinaquinasa sensor, for example vans C-2).
- Resistance genes: vanH (encoding a dehydrogenase); vanC, vanC2 / 3, vanE, vanG or vanL, encoding the ligase of type D-Ala: D-Ser. VanC them found only in Enterococcus gallinarum, and vanC2 / 3 in Enterococcus casseliflavus / flavescens, so called because some doubt that they are two species, since both have yellow pigmentation; E. flavescens would differ from E. casseliflavus for acid production from ribose. vanE and vang, only found in a small number of Enterococcus faecalis. vanC2 and vanC3 closely related loci with very few differences which are often referred vanC2 / 3.
- Accessory genes: Vant (serine racemase gene that conviertre L-serine D-serine, for example Vant called C-2); Accessory genes: VANU (transcription regulator gene vanG some type E. faecalis); vanX (D, D-dipeptidase, whose function is to hydrolyze the dimer D-Ala-D-Ala precursor); VaNy (D, D-carboxypeptidase).
- Gene of unknown function: vanW (only type vanG).
The set of genes FNPV type, with FNPV ligase, it has only been found in Paenibacillus popilliae bacteria used in biopesticides, but not found in Enterococcus spp.
Genotypic sequence of some types "van"
The order of the genes operon glycopeptide resistance of some of these types are:
- VanA Type: VanR-vans - (- vanH-vanA-vanX -) - VaNy-Vanz -
- VanB Type: vanRB-vanSB - vanYB-vanW - (- vanHB-vanB-vanXB -) -
- Vand Type: vanRD-vanSD - vanYD - (- vanHD-Vand-vanXD -) -
- FNPV type: VanR-VANS - vanYF-vanZF - (- vanHF-FNPV-vanXF -) - Vany - (only found in Paenibacillus popilliae).
- Type vanM: VanR-vans - vanYM - (- vanHM-vanM-vanXM -) -
Phenotypic sensitivity characteristics of each set of type "van"
The vanA resistance type set is characterized by high - level resistance to vancomycin (> 64 mg / mL) and teicoplanin (> 16 mg / mL), and is inducible by vancomycin both, such as teicoplanin.
The resistance vanB set type is characterized by acquired resistance to various concentrations of vancomycin (4 ? 1.024 g / mL), but not teicoplanin, and vancomycin is only inducible.
These two types, vanA and vanB, are the two most common phenotypes of resistance to glycopeptides. The genes encoding vanA and vanB phenotypes are in a transposon (Tn1546) which may be in a plasmid or on the chromosome.
The vanC1 and vanC2 / C3 types are characterized by intrinsic low - level resistance to vancomycin (2 to 32 mg / mL), and sensitivity to teicoplanin. This resistance is inducible, so that in the absence of vancomycin, vanC strains synthesize the dimer D-alanyl-D-alanine.
The vanE type found in Enterococcus faecalis is characterized by low resistance to vancomycin (16 .mu.g / ml) and sensitivity to teicoplanin (0.5 .mu.g / mL).
The Vand type is characterized by intermediate vancomycin resistance (128 ug / mL) and low level teicoplanin and constitutively expressed. The Vand genes are on the chromosome and are not transferable, so are very rare vand strains, in contrast to the large area and high prevalence of vanA and vanB strains.
The vanG type found in Enterococcus faecalis has moderate resistance to vancomycin (12-16 mg / mL) and sensibililidad teicoplanin (0.5 .mu.g / mL).
The vanM type found in studying strains possessing no vanA genes, vanB, or Vand. These strains have a set of genes called vanM. The vanM type, has high resistance to vancomycin and teicoplanin, the latter with higher MICs teicoplanin, which has the vanA type.
Tests in IVAMI
- Molecular diagnosis by amplification (PCR) for genetipos types vanA, vanB or vand, or other less frequent (vanC, vanC2 / 3, vanE, FNPV, vanG, vanL, vanM).
- Molecular diagnosis by amplification (PCR) for any type, followed by sequencing to identify the type.
- Glycopeptide resistant crop isolated.
Preservation and shipment of sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: over 2 days.
Cost of the test:
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