PCR-based detection of Klebsiella pneumoniae in blood cultures

Abstract number: 1134_04_55

Kotlovsky T., Austin L., Shalginov R., Sprecher H.

Objective:  

K. pneumonia infections are a major source of morbidity and mortality among hospitalized adult and paediatric patients. They rank second as cause of gram negative sepsis in our medical centre and 45% of them are caused by extended spectrum b-lactamase producers. Early detection is a key factor in determining the outcome of K. pneumoniae infections and numerous attempts have therefore been made during the last years to develop more rapid and accurate detection systems. The aim of the present study was to establish rapid and reliable PCR-based assays aimed at detecting gram negative bacterial DNA and K. pneumoniae nucleic acid sequences in blood cultures obtained from patients with suspected sepsis.

Methods:  

DNA was extracted from positive blood culture bottles using the benzyl alcohol-guanidine hydrochloride method. Two sets of oligonucleotide primers were designed: the first one identifies a nucleotide sequence of the 16S rRNA gene that is specific for gram negative bacteria; the second one targets K. pneumoniae haemolysin gene.

Results:  

The sensitivity of gram negative specific PCR targeted to the 3’-end of the 16S rRNA gene and to the haemolysin gene of K. pneumoniae was optimized to 100fg/ml and 200pg/ml, respectively, using DNA extracted from a standard isolate of K. pneumoniae. The specificity of the assay was established using 100 different bacterial isolates and found to be 100%. The assay performance was assessed using 108 consecutive positive blood cultures identified as part of the routine work-up of hospitalized patients. Gram staining was performed on positive blood cultures and DNA was extracted from these cultures bottles and PCR-amplified using the assay described above. The results were compared to the final microbiological diagnosis as determined using standard methodology. 108 out of 108 positive blood culture bottles were correctly identified using the PCR based assay with 100% specificity and 100% sensitivity. The turnaround time for the molecular identification was 4 hours.

Conclusions:  

PCR-based detection of K. pneumoniae directly from blood culture bottles is rapid (4h), and highly specific (100%) and sensitive (100%). Implementation of this and similar assays targeted to common bacterial pathogens should allow for earlier treatment to be provided and for a more rationale and cost-effective use of antibiotic treatment, thus cutting down costs while at the same time preventing the emergence of resistant strains.