Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
When treating an infection, it’s important that the right antimicrobial be used, both for the sake of the patient as well as for the general public. Largely because of the broad use of antimicrobials, bacteria are developing resistance to antimicrobial treatments, creating what Mayo Clinic infectious diseases specialist Pritish Tosh calls a vicious cycle: “We need to use broader and broader antimicrobials, because there are more resistant bacteria, but the more broad antimicrobials we use the more resistant bacterial there will be. … We’re already seeing situations where we have bacterial infections where there are no antimicrobials that would be predicted to treat them.”
One way to break that cycle, Tosh says, is to identify the pathogen—including its drug-resistance profile—as early as possible and use the narrowest antimicrobial treatment available, thus minimizing the opportunities for clonal expansion of resistant organisms. Here we look at the ways the medical and veterinary fields currently query antimicrobial susceptibility as well as how that might play out in the future.
Know thy enemy
When a patient presents with signs of a microbial infection, the first thing is to know if there indeed is an infection. This generally is accomplished by culturing a sample for about 24 hours, says Donna Sullivan, non-clinical professor of medicine at the University of Mississippi Medical Center. But a positive culture indicates only that a microbe is there, not what it is. So the lab then examines the cells’ morphology and performs assays such as Gram staining and catalase and coagulase tests to help identify the pathogen. A few labs have begun using MALDI-TOF mass spectrometry for bacterial classification, as well. The offending bug can then be tested against potential antimicrobials to judge its susceptibility.
An exception to this rule is the screening for methicillin-resistant Staphylococcus aureus (MRSA). “A significant amount of the population has MRSA when they come to the hospital,” and Medicaid and Medicare will not reimburse for a hospital-acquired infection, explains Sullivan, who co-authored a study using digital PCR to rapidly detect MRSA from nasal swabs without the need for prior culture [1]. Her institution, like many others, routinely screens patients for MRSA from swabs using quantitative PCR, enabling cohort and proper treatment for infected patients in a matter of hours.
But for other less-common pathogens, time from sample collection to treatment decision can take 48 to 72 hours. “Often we won’t wait until those results are back to start antimicrobials, especially if that person is very sick and requiring critical care,” says Tosh. This means starting the patient on a broad-spectrum antibiotic, or multiple antibiotics, based on educated guesses and then altering course if the pathogen is found to be resistant.
Phenotypic testing
Manual antimicrobial-susceptibility testing typically is accomplished in one of two ways. In broth-dilution tests, a culture is inoculated into serially diluted aliquots of antibiotic. After overnight incubation, the cultures are inspected for turbidity, an indicator of bacterial growth. The lowest concentration of antibiotic in which the bacteria failed to grow is termed the minimal inhibitory concentration (MIC). Panels of tests can be purchased dried or frozen in 96-well plates—for example, under the Sensititre name from Trek Diagnostic Systems (Thermo Scientific).
Many automated and partially automated systems for testing microbial sensitivity—including the BD Phoenix™, Siemens MicroScan WalkAway® plus and bioMérieux VITEK® 2 platforms—“are all based on broth microdilution and were developed using broth microdilution as a reference standard,” says Michael Dunne, vice president for research and development at bioMérieux. Such systems may offer a quicker turnaround time because of frequent monitoring and sensitive optics for detection of subtle changes in bacterial growth, and they can be directly linked to information systems.
The second manual method relies on an antimicrobial concentration gradient. Traditionally, agar plates are uniformly inoculated with the culture to be tested; onto this, commercially prepared paper discs with a fixed concentration of antibiotic are placed. The zone of growth inhibition surrounding each disk is measured and compared with standard published criteria to determine whether the isolate is susceptible, intermediate or resistant to the drug. bioMérieux’s Etest® is a variation on this theme, in which a gradient of dried antibiotic is impregnated onto the underside of a plastic strip, the top side of which has markings indicating the concentration beneath; when the strip is placed on an inoculated agar plate, the intersection of the zone of growth inhibition with the Etest indicates the MIC.
Less frequently used are tests for the presence of a particular resistance mechanism, for example an agar-based assay engendering a color change indicative of β-lactamase activity, which confers penicillin resistance.
Genotypic testing
The presence of a gene also can be used as an indicator of an organism’s drug resistance. The mecA gene confers methicillin resistance to S. aureus and is the basis for rapid MRSA qPCR tests, for example, and Mycobacteria tuberculosis resistance to rifampicin and/or isoniazid can be queried by Hain Lifescience’s GenoType MTBDR DNA hybridization assays. Both assays serve as proof of principle of a role for nucleic acid testing for antibiotic resistance.
Yet without a complete catalog of resistance factors and mechanisms—including point mutations and combinations of variations—it is impossible to test for all reasonable possibilities, even using highly multiplexed PCR. Conversely, the presence of a resistance gene does not necessarily mean that gene is intact, nor that it is expressed at levels sufficient to protect the organism. And while whole genome sequencing (WGS) and RNA-Seq can in principle reveal all the genetic and expression data required to determine treatment options, these strategies too require a comprehensive—and continuously updated—database against which sequences can be compared. (Similar arguments can be made for mass spectrometry-based predictions.) One such project, the Comprehensive Antibiotic Research Database (CARD), was announced last year [2].
Looking to the future, researchers and clinicians increasingly will be using sequence-based predictions for more actionable clinical advice, says Dunne, “but we won’t be able to do it without the phenotyping [safety] net.” So hang on to your broth and agar.
References
[1] Kelly, K, et al., “Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay,” J Clin Microbiol, 51(7):2033-9, 2013. [PubMed ID: 23596244]
[2] McArthur, AG, et al., “The Comprehensive Antibiotic Resistance Database,” Antimicrob Agents Chemother, 57(7):3348-57, 2013. [PubMed ID: 23650175]