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.
Highlights
The number of ESBLs of the CTX-M-type has increased dramatically in the past ten years, and the majority of 3rd generation cephalosporin resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae produce these enzymes [1]
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
Our results clearly showed that the distribution of the incorporated nucleotides at all nucleotide positions (NPs) was similar to that of the control strain solely harboring CTX-M-15, indicating that the presence of non-CTX-M lactamases does not disturb the efficiency of the degenerated primers (Table 4)
Summary
The number of ESBLs of the CTX-M-type has increased dramatically in the past ten years, and the majority of 3rd generation cephalosporin resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae produce these enzymes [1]. The increase of CTX-M-producers has led to an increasing use of carbapenems resulting in the emergence of carbapenem resistant Enterobacteriaceae. The CTX-M enzymes belong to class A of the serine-βlactamases and were named in reference to their uncommon preference to hydrolyze cefotaxime and cefepime more effectively than ceftazidime [2]. In the past years, CTX-M ESBLs, with highly increased hydrolyzing activity against ceftazidime (e.g. CTX-M-15), have been frequently observed in human and animal isolates as well as in environmental samples [3,4,5]. Based on the phylogenetic properties approximately 120 CTX-M variants are described so far, that cluster into five main groups [6]
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