Abstract
The functional relationship between the detection of carbapenemase activity and phenotypic resistance in Gram-negative bacterial pathogens is often ill-defined. To address this issue, we developed a two-tiered Modified Hodge Test approach for carbapenemase detection and typing, in which the use of Pseudomonas aeruginosa strain PAO1 and Escherichia coli as indicator strains conferred two levels of sensitivities to carbapenemases. When applied alongside PCR genotyping tests for existence of known carbapenemase genes in 92 carbapenem resistant clinical isolates, this method is extremely useful in elucidating the relative role by which different enzymes contributed to the prevalent carbapenem-resistance phenotypes. With this study approach, we showed that the proportion of P. aeruginosa and Acinetobacter baumannii strains whose carbapenem resistance phenotypes could at least be partially attributed to carbapenemase were 34 and 89%, respectively. Our data also facilitates detailed functional categorization of carbapenem resistance phenotypes on the basis of the types and activities of detectable carbapenemase produced by the test organism. For example, six A. baumannii isolates harboring the blaOXA-51/23-like gene without detectable enzymatic activities were identified, suggesting that other resistance mechanisms may be involved. On the other hand, there were seven P. aeruginosa strains which produced carbapenemase phenotype without harboring known carbapenemase genes, inferring the existence of some hitherto unknown resistance determinants. Findings in this work therefore provide a comprehensive view on the cellular basis of carbapenem resistance phenotypes in major Gram-negative bacterial species, paving the way for development of novel strategies to reverse the effects of the major resistance mechanisms concerned.
Highlights
Dissemination of carbapenem-resistant organisms continues to cause an increasing number of severe and often untreatable bacterial infections in nosocomial settings (Woodford et al, 2014)
Modified Hodge Test (MHT) tests were performed to assess the degree of correlation between existence of carbapenemase genes and the detection of carbapenemase activity, with results indicating that the carbapenemase blaIMP was detectable by both MHT tests, yet the blaKPC enzyme was detectable by MHTPAO1 only (Figure 1)
False positives have been described in MHT to detect carbapenemases in P. aeruginosa previously, it can not rule out the possibility that other unknown carbapenem resistance mechanisms are present in these seven strains (Vasoo et al, 2013)
Summary
Dissemination of carbapenem-resistant organisms continues to cause an increasing number of severe and often untreatable bacterial infections in nosocomial settings (Woodford et al, 2014). OXA-51 has been reported to be intrinsic to Acinetobacter baumannii and it normally does not lead to carbapenem resistance unless an Insertion Sequence, ISAba, is introduced into its upstream region, enhancing the strength of the promoter and causing over-expression of the oxa-51 gene Even if this happens, it has been reported that the CHDL OXA-51 produced exhibits only weak carbapenemase activity (Turton et al, 2006). We showed that combined analysis of results of carbapenemase detection tests with MHT and PAO1– MHT, which cover detection sensitivity toward CHDLs and other carbapenemases, as well as those of PCR genotyping of carbapenemase genes, allows highly accurate assessment of the type and relative role of the carbapenemase involved in conferring the observable phenotype This analytical approach shall help better define the molecular basis of clinical carbapenem resistance and establish a profile of carbapenemase-encoding elements commonly harbored by each of the key Gram-negative pathogens. Such data are essential for future development of novel antimicrobial strategies, especially those involved in carbapenemase inhibition
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