Volume 39 Number 3 | June 2025
The Time is PAST—the FUTURE is NOW!
Pat Tille, PhD, MLS(ASCP), AHI(AMT), FACSc, ASCLS President
Over the past decade, we have witnessed the influx of molecular diagnostics and the identification of organisms using genetic sequences in place of phenotypic characteristics. Not only has this provided a means to rapidly identify organisms, but it has also introduced new organisms and required the reclassification and naming of many others.
One of the most notable name changes is Clostridium difficile to Clostridiodes difficile. Others are not so common in the laboratory, but maybe more evident in education. The broad fish tapeworm, Diphylobothrium latum is now Dibothriocephalus latus. Although some would argue that names are insignificant, they are the cornerstone of taxonomic classification.
Molecular identification provides a rapid method for diagnosis and treatment improving patient outcomes. But more important than identification of the organism, is the characterization of antibiotic resistance mechanisms. In 2019 alone, there was an estimated 1.29 million deaths due to antimicrobial resistance.1
Another notable example is the reclassification of the gram-negative enteric organism, Enterobacteriaceae to the Order Enterobacterales containing five families. But does reorganizing and renaming bacteria solve the problem?
Escherichia coli, a common organism that resides in the human gastrointestinal tract as normal flora, is also a notable pathogen. E. coli has six different pathotypes that are considered diarrheagenic. Each of the specific pathotypes carry various virulence genes.
E. coli is an example of an organism where knowing the identification is not enough to properly manage and treat a patient. The variation seen in E. coli is due to the ability to alter gene expression by deleting sequences or genes, acquiring new genes, or simply turning genes on and off in response to the environment. In some cases, a simple phenotypic test such as the lack of sorbitol fermentation on MacConkey sorbitol agar by E. coli O157-H7 in comparison to other strains.
However, traditional phenotypic methods do not provide information beyond the identification of the bacteria in most cases that permit the differentiation of each pathotype. This traditional test also requires growth of the organism for a period of 18-24 hours before this reaction is evident. Molecular diagnostics can be used to rapidly identify E. coli O157-H7 using unique genetic markers that include the open reading frame (ORFZ3276), the shiga toxin (Stx), the adherence gene intimin (eae), and hemolysin genes (hly).2
E. coli is but one example of an organism that may look harmless upon initial identification but can harbor multiple significant virulence genes that can cause serious human disease. Other organisms include carbapenemase (KPC)-producing Klebsiella pneumoniae, CTX-M-15-producing E. coli, and OXA-23-producing Acinetobacter baumannii.1 These organisms, as well as others, obtain the antibiotic resistance genes from mobile genetic elements (MGE).
A single organism may harbor many MGEs that can be classified as the bacteria’s mobilome. These mobile genetic elements include plasmids, integrons, and transposons. Transposons are small genetic elements that contain the enzyme transposase that functions to excise the element from a nucleic acid molecule, such as a plasmid or chromosome, and move it to another where it can reintegrate into the element. Transposons may be simple, known as insertion sequences that carry no accessory genes, or they may be complex or compound and consist of two insertion sequences that flank an antimicrobial resistant gene that can be carried from one nucleic acid molecule to another.
Integrons are more complex than transposons and carry the integrase gene, which promotes integration into different molecules such as plasmids or chromosomes. Integrons often carry larger genetic sequences or cassettes of antimicrobial resistance genes that may or may not include a promoter region that allows for transcription of the gene upon integration into a chromosome.
Plasmids, in comparison to transposons and integrons, can carry many different genes from one organism to another, along with carrying multiple MGEs. In some cases, the MGE or plasmid, is self-replicating and can increase the copy number of virulence genes to facilitate transfer to other bacteria. This can occur vertically from one type of organism to another, or simply horizontally through bacterial division and growth.1,2
Transfer of MGEs knows no boundaries and is an ongoing challenge in the treatment of serious infections. Phenotypic testing and detection of antimicrobial resistance can take two to three days. This is troublesome in severe cases of sepsis on critically ill patients. Combination molecular panels that can detect specific markers and pathogens simultaneously are essential in ensuring rapid diagnosis and critical care in this situation. The panels are most often based on polymerase chain reaction (PCR) or amplification using a multiplex system. Although Next-Generation Sequencing (NGS) is on the rise, it has yet to become a staple in the routine clinical microbiology laboratory.
Although molecular diagnostics in clinical microbiology seems to be the best way to address the rapid transmission rate of multidrug resistant organisms and the ability to identify and treat the infections, it is not without limitations. There is a significant financial investment in instrumentation, supplies, and training to implement a new molecular system in a laboratory. With advanced methods such as NGS, bioinformatics and data analysis also become a challenge. Large public databases are available with gene sequences and AMR markers, but the information is inconsistent. Standardization is needed and ways to verify that any information archived is accurate.
With the high cost and need for more data, is cost a barrier the laboratory can afford? How long must the laboratory sacrifice quality care and the increase in emerging antibiotic resistance organisms associated with patient morbidity and mortality? Although there are limitations to implementing molecular diagnostic techniques, it is a battle the laboratory must wage to continue to improve databases and slow the spread of antibiotic resistance while improving treatment and infection control and prevention.
As molecular diagnostic methods become more streamlined and available in multiplex PCR closed systems, this should lead to more data to reinforce the need to continue the exploration of NGS and whole genome sequencing as not something in the future, but a necessary step in the evolution of laboratory diagnostics.
References:
- Fahy S, O’Connor JA, Sleator RD, Lucey B. From Species to Genes: A New Diagnostic Paradigm. Antibiotics (Basel). 2024 Jul 17;13(7):661. doi: 10.3390/antibiotics13070661. PMID: 39061343; PMCID: PMC11274079.
- Tille PM. Bailey and Scotts Diagnostic Microbiology, 16th Edition, Elsevier, St. Louise MO, 2026.
Pat Tille is the Graduate Program Director and Professor at the University of Cincinnati in Cincinnati, Ohio.