Diagn Microbiol Infect Dis. 2013 Oct;77(2):113-7.

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens.

Geyer CN, Hanson ND.

Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology (C.R.A.B), 2500 California Plaza, Omaha, NE 68178, USA.



A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.

KEYWORDS: Beta-lactamase genes, Diagnostic PCR, Rapid amplification

PMID: 23891223



It is no exaggeration to say that antibiotic resistant organisms are a global infectious disease crisis that needs immediate attention. This is especially true for Gram-negative organisms such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., and Pseudomonas aeruginosa where, until recently, the resistance problem has been under appreciated by the medical field. While clinical microbiologists have been concerned about the emergence of resistance in these organisms for more than 2 decades, the complex mechanisms by which resistance occurs has been difficult to assess by traditional clinical laboratory procedures. Clinical microbiologists could identify the occurrence and increased frequency of resistance but advances in methodologies for recognizing the mechanisms of resistance in the clinical laboratory were slow to come (Livermore 2012). The result has been a worsening of the resistance problem resulting in a global crisis that is affecting infection control measures and therapeutic treatment options in thousands of hospitals worldwide.

Over the last 15 years our laboratory has sought to develop rapid molecular (i.e., PCR-based) protocols for resistance detection that could supplement the standard laboratory techniques used in the clinical microbiology laboratory (Perez 2002, Pitout 2004, Geyer 2012, 2014, and Roth 2012). We focused on the identification of beta-lactamase genes for which the resistance phenotype can be difficult to detect using traditional approaches. In recent years, β-lactamase genes encoding the CTX-M extended-spectrum β-lactamases, plasmid-encoded AmpC β-lactamases, and carbapenemases such as KPC, NDM, VIM, IMP, and OXA-48 have been the focus of our assay development. Recently the Centers for Disease Control described the threat level of organisms that produced these enzymes as urgent, serious, and concerning, underscoring the importance of novel diagnostic procedures for their identification. While other approaches are being developed to detect these resistance mechanisms (Dortet et al. 2012, Kim et al. 2007) PCR is a well-established methodology to which most laboratories have access. In recent years laboratories with access to PCR amplification have attempted to develop “home brew” methods for identifying some of these resistance genes. However, laboratories typically do not have the personnel or resources to develop such assays and would clearly benefit from optimized protocols to use and validate in the clinical setting. Large and small laboratories alike are struggling with Gram-negative resistance issues. Therefore, there is a need for a variety of both phenotypic and molecular diagnostic methods allowing labs to pick the approach that best suits their needs. The rapid amplification technology described in our DMID paper allows for rapid detection of the resistance gene from a pure culture in less than two hours. For patient therapy, there is obvious value in quickly identifying the resistance mechanism(s) contributing to what will ultimately be a susceptibility pattern, helping to guide selection of the most appropriate antibiotic in a short time frame. Expedited identification of the most appropriate antibiotic for the patient is clearly beneficial for recovery (Quresh et al. 2012). The ability of reference laboratories to rapidly identify pathogen/resistance mechanisms is imperative in assisting hospitals to recognize infection control problems and expedite infection control and patient isolation procedures.

At the present time molecular detection is being used mainly for infection control issues in hospital settings and not so much in determining therapeutic options, although the exception may be the identification of carbapenemase genes, especially KPC. However, in the future it will be important to initiate clinical trials using molecular-based assays to determine the impact these types of diagnostic tests have on therapeutic outcome. Until those data are collected some laboratories will embrace the molecular technology to identify resistance mechanisms and thus understand the selective pressures driving or sustaining resistant pathogens in the patient population. However, other laboratories will decide to stay with the status quo arguing that there is little or no published clinical evidence justifying the need for molecular-based assays in the evaluation of therapeutic options. Unfortunately, clinical decisions not including molecular diagnostics for the detection of resistance mechanisms in Gram-negative pathogens continue to only benefit the pathogen thus jeopardizing the healthcare of the patient.



Dortet, L., L. Poirel, and P. Nordman. 2012. Rapid identification of carbapenemase types in Enterobacteriaceae and Pseudomonas spp. by using a biochemical test. Antimicrob. Agents Chemother. 56:6437-6440.

Geyer, C., M. Reisbig, and N. D. Hanson. 2012. Development of a TaqMan Multiplex PCR Assay for Detection of Plasmid-Mediated AmpC β-Lactamase Genes. J. Clin. Microbiol. 50: 3722–3725.

Geyer, C. and N. D. Hanson. 2014. Multiplex High-Resolution Melting Analysis as a Diagnostic Tool for Detection of Plasmid-Mediated AmpC β-Lactamase. J. Clin. Microbiol. 52: 1262–1265.

Kim, S-Y, S. G. Hong, E. S. Moland, and K. S. Thomoson. Convenient test using a combination of chelating agents for detection of metallo-β-lactamases in the clinical laboratory. J. Clin. Microbiol. 45:2798-2801.

Livermore, D. M., J. M. Andrews, P. M. Hawkey, P-L Ho, Y. Keness, Y. Doi, D. Paterson, and Neil Woodford. 2012. Are susceptibility tests enough, or should laboratories still seek ESBLs and carbapenemases directly? J. Antimicrob. Chemother. 67: 1569-1577.

Perez-Perez, J. and N. D. Hanson. 2002. Detection of Plasmid-Mediated AmpC β-Lactamase Genes in

Clinical Isolates by Using Multiplex PCR. J. Clin. Microbiol. 40: 2153–2162.

Pitout, J., A. Hossain, N. D. Hanson. 2004. Phenotypic and Molecular Detection of CTX-M-β-Lactamases Produced by Escherichia coli and Klebsiella spp. J. Clin. Microbiol. 42: 5715–5721.

Roth, A. and N. D. Hanson. 2013. Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses. J. Clin. Microbiol. 51:61-65.

Qureshi, Z.A., D. L. Paterson, A.Y, Peleg, J.M. Adams-Haduch, K. A. Shutt, D. L. Pakstis, E. Sordillo, B. Polsky, G. Sandkovsky, M. K. Bhussar, and Y. Doi. 2012. Clinical characteristics of bacteremia caused by extended-spectrum β-lactamase-producing Enterobacteriaceae in the era of CTX-M-type and KPC-type β-lactamases. Clin. Microbiol Infect 18: 887-893.

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