Product Spotlight: Streck's Rapid Multiplex PCR Kits for the ID of β-Lactamase Genes


December 2015 - Vol.4 No. 8 - Page #20

As the number of infections caused by antibiotic resistant pathogens has increased over the past few decades, treatment options have decreased, resulting in increased morbidity and mortality of infected patients. The Centers for Disease Control and Prevention (CDC) and the World Health Organization recognize the threat these organisms pose to public health.1 In fact, the CDC assembled a list of the most problematic resistant bacterial pathogens, which includes carbapenem-resistant Enterobacteriaceae, extended-spectrum β-lactamase (ESBL)-producing Gram-negative organisms, and multidrug resistant Pseudomonas aeruginosa and Acinetobacter spp.2 Although methicillin-resistant Staphylococcus aureus (MRSA) still is a significant issue in hospital settings, antibiotic treatment options are available. The major challenge, therefore, lies with treating infections caused by resistant Gram-negative organisms. Although new drugs are emerging to treat these infections, their numbers are small, and they are not yet used widely. When drug options are limited, health care workers must rely more heavily on avenues of prevention, including antibiotic stewardship, infection control, and identification of resistance mechanisms so that physicians can prescribe the antibiotic(s) most likely to yield success.3

Identifying Mechanisms of Resistance
The use of molecular diagnostics has gained acceptance in recent years as a method for identifying the mechanisms of resistance in some bacterial infections occurring in hospital and community settings. Although β-lactam antibiotics are widely available and constitute the largest class of antibiotics, enzymes known as β-lactamases that are produced by certain bacteria can render these drugs ineffective. Phenotypic susceptibility testing can identify the antibiotics to which the organism may be resistant, but such testing cannot determine the specific mechanism of resistance. Over 1,000 different types of β-lactamases exist with some more clinically relevant than others. Many Gram-negative organisms in the family Enterobacteriaceae harbor β-lactamase genes on mobile genetic elements that allow these genes to be transferred to different genera and species of bacteria. In fact, these multidrug resistant pathogens can carry up to eight different β-lactamases within one organism.4 The spread of these organisms and/or the genes that encode these β-lactamases can influence infection rates in hospitals, the effectiveness of antibiotic stewardship programs, and the mortality rate of infected patients.

Surveillance of resistant organisms is the key to combating the spread of multidrug resistant pathogens and keeping them from becoming endemic. The complexity of β-lactamase-producing pathogens warrants not only susceptibility testing, but also a molecular evaluation for the presence of the genes encoding the β-lactamases indicated as major threats by the CDC. These include those genes encoding carbapenemases, extended-spectrum β-lactamases, and plasmid-encoding ampC β-lactamases.5 The carbapenemases that most concern clinical microbiologists and physicians include members of the KPC, NDM, IMP, VIM, and OXA-48 families. Although there are hundreds of ESBLs, the most concerning ones are those belonging to the CTX-M family. Two distinct family members from this large family of β-lactamases, CTX-M-14 and CTX-M-15, are the most prevalent ESBLs worldwide. Organisms that produce these enzymes are both hospital– and community-acquired. The genes for these types of enzymes are carried mainly by Escherichia coli and are highly associated with urinary tract infections.

The other group of enzymes that is of major concern is the plasmid-encoded ampC β-lactamases. There are no Clinical and Laboratory Standards Institute (CLSI) guidelines for phenotypically evaluating the susceptibility of plasmid-encoded ampCs. As a result, these enzymes often are misidentified as ESBLs or phenotypically found susceptible to third-generation cephalosporins. Such misidentification can result in an inappropriate β-lactam antibiotic being selected for treatment, which can lead to therapeutic failure and possible death for the patient.6

Two Identification Kits
Streck recently developed two multiplex PCR kits for the rapid identification of clinically relevant β-lactamase genes. Using one PCR reaction, the ARM-D for β-Lactamase ID kit detects nine different β-lactamase gene families: CMY, DHA, CTX-M-14, VIM, NDM, IMP, KPC, CTX-M-15, and OXA-48. Similarly, the Philisa ampC ID kit identifies six plasmid-associated ampC resistance genes. The kits offer several advantages:

  • Both incorporate an internal control during PCR amplification to validate successful extraction of bacterial DNA, thereby eliminating false-negative results due to poor template preparation.
  • The kits combine control templates into two tubes, which can act as size markers and as verification of a successful PCR reaction.
  • All reagents required for the PCR are provided within the ARM-D for β-Lactamase ID kit. It contains a master mix consisting of enzyme, dNTPs, buffer, and a primer mix for all primers needed for the multiplex reaction. The only thing required from the testing laboratory is template. Similarly, the Philisa ampC ID kit contains everything necessary to run the reactions except the enzyme. The PhilisaFAST polymerase is sold separately. Alternatively, other enzymes can be used in the assay.

The end-point reactions of both assays have been validated using two types of thermal cyclers. The Philisa is a rapid amplification thermal cycler available from Streck. Using these kits and the Philisa System, the PCR reactions can be completed in approximately 20 minutes. Amplification reactions also can be performed using any other thermal cycler. Agarose gel electrophoresis is required for separation of PCR products and visualization by staining. The entire process takes less than 3 hours.

Incorporating Assays into Lab Workflow
How these assays are incorporated into the workflow of the clinical laboratory depends on the individual hospital. If antibiograms indicate high numbers of organisms producing ESBLs/ampCs/carbapenemases, lab staff can run these PCR assays at the same time as susceptibility assays to determine resistant genotypes prior to obtaining susceptibility results. This can yield information important for choosing antibiotic therapy. For example, a positive KPC result, which has been shown to be associated with higher rates of morbidity and mortality in patients,7 may point physicians toward a different course of treatment than a negative result.

Alternatively, the PCR assay can be performed after the susceptibility profile is evaluated and found to indicate resistance to third-generation cephalosporins, cefoxitin, or carbapenems (see FIGURE 1). If the number of resistant organisms in the hospital is low, then running assays for infection control purposes monthly is probably sufficient.

Surveillance is crucial to understanding and addressing resistant pathogens. Because no two hospitals are the same, it is up to each facility to determine the best approach for incorporating this molecular testing into the lab’s operation. Ultimately, this testing will generate valuable information to help guide infection control practices, antibiotic stewardship, and the selection of antibiotic therapy.

References

  1. Perez F, Villegas MV. The role of surveillance systems in confronting the global crisis of antibiotic-resistant bacteria. Curr Opin Infect Dis. 2015;28(4):375-383.
  2. Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013. http://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf. Accessed July 20, 2015.
  3. Amin AN, Deruelle D. Healthcare-associated infections, infection control and the potential of new antibiotics in development in the USA. Future Microbiol. 2015;10:1049-1062.
  4. Moland ES, Hong SG, Thomson KS, et al. Klebsiella pneumoniae isolate producing at least eight different β-lactamases, including AmpC and KPC β-lactamases. Antimicrob Agents Chemother. 2007;51(2):800-801.
  5. Hanson ND. Molecular Diagnostics Could Help in Coping with Hidden β-Lactamases. Microbe. 2010;5:333-339.
  6. Pai H, Kang CI, Byeon JH, et al. Epidemiology and clinical features of bloodstream infections caused by AmpC-Type-β-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother. 2004;48(10):3720-3728.
  7. Tumbarello M, Trecarichi EM, De Rosa FG, et al. Infections caused by KPC-producing Klebsiella pneumoniae: differences in therapy and mortality in a multicentre study. J Antimicrob Chemother. 2015;70(7):2133–2143.

Nancy D. Hanson, PhD, is a professor in the department of medical microbiology and immunology at Creighton University, Omaha, Nebraska, and director of the center for anti-infectives and biotechnology.

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