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Bologna Researchers Use MALDI Biotyper to Identify Carbapenem-Resistant Bacteria

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NEW YORK (GenomeWeb) – Researchers from the University of Bologna and Bruker have used Bruker's MALDI Biotyper to identify antibiotic resistance in clinical microbiology specimens.

In a study published last month in the Journal of Clinical Microbiology, the researchers managed with the Biotyper to detect Klebsiella pneumoniae strains containing the Klebsiella pneumoniae carbapenemase (KPC) protein, which can confer resistance to carbapenems.

Carbapenems are commonly used in hospitals in patients with infections already resistant to other antibiotics, and, as such, present a significant challenge for clinicians. KPC-mediated resistance is widespread across Italy, Paolo Gaibani, a researcher at Bologna's S. Orsola-Malpighi University Hospital and first author on the JCM paper, told GenomeWeb.

"This is a big problem, because this type of resistance is very common in [Italy]," he said, adding that the MALDI-based detection method could offer improvements in speed, cost, and ease of use compared to existing approaches like phenotypic testing and PCR.

To identify potential markers, Gaibani and his colleagues compared protein spectra from KPC-containing strains and those without KPC, identifying a total of 10 peaks that appeared to distinguish between the two populations. Further evaluation of these peaks found that all but one, a peak at 11.109 Da, were present in both strains with and without KPC. The 11.109 Da peak was present in 30 of 24 KPC-producing K.pneumoniae strains and was absent in all KPC-negative samples, indicating that it could be a suitable marker for KPC presence.

They then tested the marker in an additional 340 KPC-positive isolates and 214 KPC-negative isolates, finding that the Biotyper detected the 11.109 Da peak in 98 percent of the KPC-positive samples (333 out of 340) and failed to detect it in 99 percent of the KPC-negative samples (212 out of 214). Overall, the method detected KPC-positive isolates with 98 percent sensitivity, 99 percent specificity, and a positive predictive value of 99 percent.

The Italian researchers' work follows that of a National Institutes of Health team that initially identified the link between the 11.109 Da peak, which identifies the pKpQIL_p019 protein, and the bkaKPC gene, which produces the KPC protein, in a 2014 JCM paper.

The NIH researchers followed up that work with another JCM paper in 2015 in which they tested whether detection of the pKpQIL_p019 protein via the MALDI Biotyper could identify carbapenem resistance in a blinded analysis of 140 previously characterized Enterobacteriaceae representing eight genera and at least 14 different species along with a retrospective analysis of 720 clinical Enterobacteriaceae spectra.

In the 140 Enterobacteriaceae, the NIH researchers found that all 26 isolates that were positive for p019 also contained blaKPC and were carbapenem resistant. Additionally, the MALDI assay did not detect p019 in any isolates where the presence of the gene had not been confirmed by PCR.

In the retrospective analysis, they detected p019 in nine of the 720 spectra, seven of which were actually from p019-containing, carbapenem-resistant isolates.

The Italian JCM study provides additional evidence of the usefulness of this method.

KPC provides resistance by hydrolyzing carbapenemases, and other researchers are developing methods using MALDI mass spec to identify resistant strains by detecting the hydrolysis products generated by KPC. The advantage of the p019-based approach is that researchers detect the presence or absence of this protein through the spectra collected as part of routine MALDI identification workflows.

The p019-based method does not detect KPC directly, however. Rather, the genes for p019 and KPC are frequently expressed on the same plasmid, meaning that p019 can be used as a proxy for the presence of KPC.

That means that resistance in bacteria with the KPC gene but not the p019 gene will be missed by the method. For instance, in the 140-sample set used by the NIH researchers, only 26 isolates were positive for p019 (which the researchers confirmed via PCR),  but the set contained a total of 86 carbapenem-resistant isolates.

To better assess the applicability of the approach, Gaibani and his colleagues looked for the presence of p019 across a variety of different KPC-containing plasmids, analyzing the whole genomes of 159 KPC-containing K. pneumoniae strains from a variety of countries. This analysis found that p019 was present in 135 of 138 organisms containing a plasmid with the Tn4401a transposon but absent in 20 organisms with the Tn4401b transposon.

The fact that p019 is not always present with KPC is a significant limitation for the MALDI-based approach, but Gaibani and his co-authors noted that the high positive predictive value of the test indicates it can be useful for initial screens for carbapenem resistance, with negative results passed on for additional confirmation.

Indeed, the NIH researchers have instituted a script that automatically searches their clinical MALDI spectra for the presence of p019 that they use routinely at the NIH Clinical Center. Gaibani said that he and his colleagues are currently validating the approach against existing methods with the aim of incorporating it into their clinical practice.

Currently, they use phenotypic testing along with confirmation of positive results by PCR, but the MALDI approach offers a faster and simpler alternative.

Bruker's MALDI Biotyper and BioMérieux's Vitek MS systems have made significant inroads in the research and clinical microbiology markets for organism identification. More recently, MALDI-based methods for detecting antibiotic resistance have become an area of interest for both companies, as is indicated by Bruker's participation in the Italian JCM study.

In addition to the p019-based approach, Bruker has developed a research-use-only method on the MALDI Biotyper for detection of carbapenem-resistant strains of Bacteroides fragilis and Staphylococcus aureus via detection of the presence of, respectively, the cfiA gene and the mecA cassette expressing the protein PSM.

The company also this year released its MBT STAR-BL assay, which detects antibiotic resistance in Gram-negative bacteria by looking for products generated when these antibiotics are degraded by carbapenemases.

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