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Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).

Stein C, Makarewicz O, Pfeifer Y, Brandt C, Ramos JC, Klinger M, Pletz MW - PLoS ONE (2013)

Bottom Line: Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively.Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers.The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Infection's Control, Jena University Hospital, Jena, Germany.

ABSTRACT
The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.

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Representative pyrograms of CTX-M-1.Sequence obtained by degenerated primer seqCTX-M211 (A) and seqCTX-M705 (B). The sequentially applied nucleotides are given at the X-axis, alternative NPs are boxed and the bases are indicated on the top. The Y-axis shows the relative signal intensity. The probabilities of the nucleotides were calculated by the PyroMark-evaluation software in percent.
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pone-0080079-g003: Representative pyrograms of CTX-M-1.Sequence obtained by degenerated primer seqCTX-M211 (A) and seqCTX-M705 (B). The sequentially applied nucleotides are given at the X-axis, alternative NPs are boxed and the bases are indicated on the top. The Y-axis shows the relative signal intensity. The probabilities of the nucleotides were calculated by the PyroMark-evaluation software in percent.

Mentions: In general, protocols for pyrosequencing are usually designed for determination of only one or few substitutions within a short sequence. However, in many CTX-M variants, substitutions are distributed over the entire gene. For the chosen discriminating region, ranging from +227 to +246 (Figure 1), we developed an optimized sequential application of nucleotides including an unspecific adenine at nucleotide position (NP) 4 that allowed a clear resolution of this position bearing a high GC-content (for details see Figure 3). The sequencing primer seqCTX-M211 was also designed as a degenerated oligonucleotide. The read outs were evaluated by the PyroMark Assay Design 2.0. Ink software. The probabilities of the incorporated nucleotides at the positions of interest are displayed as percentages (see Table 2). Specific distribution patterns could be allocated to the individual groups: Cytosine at NP 3 and NP 6 with an evidence of >90 % was unique for group 1 members. Thymine at NP 4 differentiates the clinically relevant CTX-M-1 and -32 and further variants (114, 116, 23, 52, 53, 55, 57, 60, 61, 69, and 79) from the other representatives of group 1. Groups 8 and 25 showed unique distributions for guanine at NP 3 and NP 6, simultaneously differing at NP 1 with guanine for group 8 (100 %) and adenine for group 25 (100 %). Variants of group 9 could be identified by the thymine at NP 2 since group 1, 2, 8, and 25 exhibited strong preferences to guanine (>94 %). Group 2 could be distinguished from other groups by the presence of guanine at NP 1 and NP 5, even when the evidence was only 76 % or 89 %, respectively. Guanine at NP 5 separated group 2 from group 8 (adenine).


Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).

Stein C, Makarewicz O, Pfeifer Y, Brandt C, Ramos JC, Klinger M, Pletz MW - PLoS ONE (2013)

Representative pyrograms of CTX-M-1.Sequence obtained by degenerated primer seqCTX-M211 (A) and seqCTX-M705 (B). The sequentially applied nucleotides are given at the X-axis, alternative NPs are boxed and the bases are indicated on the top. The Y-axis shows the relative signal intensity. The probabilities of the nucleotides were calculated by the PyroMark-evaluation software in percent.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818264&req=5

pone-0080079-g003: Representative pyrograms of CTX-M-1.Sequence obtained by degenerated primer seqCTX-M211 (A) and seqCTX-M705 (B). The sequentially applied nucleotides are given at the X-axis, alternative NPs are boxed and the bases are indicated on the top. The Y-axis shows the relative signal intensity. The probabilities of the nucleotides were calculated by the PyroMark-evaluation software in percent.
Mentions: In general, protocols for pyrosequencing are usually designed for determination of only one or few substitutions within a short sequence. However, in many CTX-M variants, substitutions are distributed over the entire gene. For the chosen discriminating region, ranging from +227 to +246 (Figure 1), we developed an optimized sequential application of nucleotides including an unspecific adenine at nucleotide position (NP) 4 that allowed a clear resolution of this position bearing a high GC-content (for details see Figure 3). The sequencing primer seqCTX-M211 was also designed as a degenerated oligonucleotide. The read outs were evaluated by the PyroMark Assay Design 2.0. Ink software. The probabilities of the incorporated nucleotides at the positions of interest are displayed as percentages (see Table 2). Specific distribution patterns could be allocated to the individual groups: Cytosine at NP 3 and NP 6 with an evidence of >90 % was unique for group 1 members. Thymine at NP 4 differentiates the clinically relevant CTX-M-1 and -32 and further variants (114, 116, 23, 52, 53, 55, 57, 60, 61, 69, and 79) from the other representatives of group 1. Groups 8 and 25 showed unique distributions for guanine at NP 3 and NP 6, simultaneously differing at NP 1 with guanine for group 8 (100 %) and adenine for group 25 (100 %). Variants of group 9 could be identified by the thymine at NP 2 since group 1, 2, 8, and 25 exhibited strong preferences to guanine (>94 %). Group 2 could be distinguished from other groups by the presence of guanine at NP 1 and NP 5, even when the evidence was only 76 % or 89 %, respectively. Guanine at NP 5 separated group 2 from group 8 (adenine).

Bottom Line: Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively.Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers.The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Infection's Control, Jena University Hospital, Jena, Germany.

ABSTRACT
The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.

Show MeSH
Related in: MedlinePlus