Biological Standards in Life Sciences – Enhancing Reproducibility

Cara N. Wilder, Ph.D.

With tremendous breakthroughs being made in the life sciences every day, it is critical that reported and published data are not only reliable and accurate, but reproducible as well. Unfortunately, with the inherent variability of biological materials and reagents, as well as the differences in analytical techniques and data reporting, irreproducibility is still a universal problem throughout both commercial and academic settings. In fact, this issue has resulted in significant, long-lasting effects including extensive losses in time and funding, perpetuation of false data, reputational damage, and impairment of professional relationships and collaborations. Here, we will briefly discuss how variations in biological materials can affect reproducibility and how the use of organisms as standards can help counteract these effects.

 Within the life sciences, irreproducibility can be caused by a number of underlying factors ranging from the biological materials and reagents used for a set of experiments to how the experiment is performed and analyzed. For instance, bacteria can vary quite a bit at the species level, which is why we often see further characterization at the subspecies and strain level. Within any given bacterial species, representative strains will exhibit similar phenotypic and genotypic traits, such as characteristic morphologies, similar metabolic requirements, and conserved ribosomal 16S sequences. However, as similar as these strains may be, selective and societal pressures can lead to genetic mutations or the acquisition of laterally transferred genetic elements that may result in significant phenotypic changes such as variations in serotype, intracellular signaling, protein expression, pathogenicity, or drug-resistance. In turn, this can affect experimental reproducibility between research facilities that are not using the same variant. Overall, the inherent differences of biological materials bring unique challenges to establishing reliable assays.

To help control for the intrinsic differences between the strains used within the life sciences, and thus enhance experimental reproducibility, the use of biological standards is recommended. Biological standards are defined as well-characterized, authenticated, purified biological reference materials – qualities of which are essential for their effective use in assay validation and calibration, research and development, diagnostics, etc. For example, when testing consumable or pharmaceutical products for specific microbial pathogens, the use of appropriate biological reference materials can ensure that the assay is sensitive and precise enough to detect the presence of objectionable microbial contaminants. The use of biological standards is equally important in clinical settings for the detection and identification of infectious agents. As you can image, the sensitivity and specificity of these assay can have a profound effect on public health.

Currently, biological standards are developed and produced by a number of entities, including government agencies, commercial companies, and non-profit institutions. ATCC, for example, produces both animal cell lines and microorganisms as Certified Reference Materials (CRMs). These biological standards are produced under an ISO Guide 34:2009 accreditation -- a process that offers confirmed identity, well-defined characteristics, and an established chain of custody. Moreover, ATCC CRMs are stable with respect to one or more specified properties, which makes them ideal for performing in challenge assays, verifying or comparing test methods, and benchmarking critical assay performance during assay validation or implementation.

Overall, the inherent variability of biological materials can significantly affect the quality and reproducibility of data. Through the use of standardized biological reference materials, assay consistency and accuracy can be improved.

Eradicating Helicobacter pylori infection

Cara N. Wilder, Ph.D.

Helicobacter pylori is a Gram-negative, microaerophilic bacterium known to inhabit the stomach lining of at least 50% of the human population. This pathogen is transmitted to humans through the consumption of contaminated food and water, as well as through direct contact with infected individuals. Once ingested, H. pylori will colonize the surface of stomach epithelial cells, resulting in either asymptomatic carriage or complications including chronic gastritis, peptic ulcers, or stomach cancer.

The most common treatment of H. pylori infections is through the use of a triple regimen that combines two antibiotics (clarithromycin, amoxicillin) and a proton pump inhibitor (PPI). Though this treatment is effective in most patients, recent reports have indicated that successful bacterial eradication is decreasing due to the emergence of clarithromycin-resistant strains. To aid in the treatment of these antibiotic-resistant infections, many clinicians have turned toward the use of second-line treatments such as a bismuth-containing quadruple therapy (EBMT) or a moxifloxacin-containing triple therapy (MEA). However, little is known regarding the efficacy of these second-line therapies.

In a recent study, Kim et al. sought to evaluate the rate of H. pylori reinfection following EBMT or MEA treatment. In this analysis, 648 patients who failed bacterial eradication through the standard triple therapy were treated with either the EBMT or the MEA second-line therapies. At four weeks following treatment, patients were examined for H. pylori colonization through either the ¹³C urea breath test or by invasive analysis. From this investigation, the annual reinfection rate from EBMT and MEA treatment was found to be 4.45% and 6.46%, respectively. Overall, the long-term reinfection rate of H. pylori remained low following both second-line treatments, suggesting that there is no significant evidence that reinfection of H. pylori is related to the eradication program.

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Prevention of Toxoplasmosis

Histopathology of active toxoplasmosis of myocardium.
Photo courtesy of  EP Ewing Jr. and CDC

Cara N. Wilder, Ph.D.

Toxoplasma gondii is a ubiquitous obligate, intracellular parasitic protozoan known to cause toxoplasmosis in a number of warm-blooded animals, including humans. This protist is transmitted to humans through the consumption of undercooked meat from infected animals, the ingestion of food or water contaminated with oocytes from infected cat feces, or by transplacental transmission. In healthy individuals, toxoplasmosis is relatively asymptomatic and self-limiting. However, this illness can silently affect pregnant women and result in severe consequences for the fetus including neurological impairment, chorioretinitis, or death. T. gondii can also affect immune-compromised individuals, resulting in cerebral or extra-cerebral toxoplasmosis.

Currently, the CDC considers toxoplasmosis to be one of the leading causes of death attributed to foodborne illness. Moreover, T. gondii infection in domestic animals represents a significant economic and public health threat due to the potential for foodborne outbreaks. Unfortunately, treatment of toxoplasmosis is difficult due to both the severe side-effects of the drug as well as the potential for re-infection. Thus, the development of effective preventative treatments is of great importance.

In recent study, Wang et al. analyzed the protective efficacy of recombinant T. gondii protein disulfide isomerase (PDI) as a potential target for the development of a novel vaccine. This particular antigen was chosen as a candidate vaccine target as it is soluble, demonstrates conserved homology among the three distinct clonal lineages of T. gondii strains, and is highly expressed on the outer surface of T. gondii tachyzoites. In this study, BALB/c mice were intranasally immunized with varying concentrations of recombinant T. gondii PDI (rTgPDI), and the resulting immunological response was evaluated by lymphoproliferative assays and by cytokine and antibody measurements. In addition to this analysis, immunized mice were also challenged with tachyzoites from T. gondii strain RH. Following this challenge, the survival time of the mice was assessed and the number of brain and liver tachyzoites enumerated.

From these analyses, the group discovered that immunization with 30 µg of rTgPDI demonstrated higher levels of anti-PDI antibody production, a strong lymphoproliferative response, and high levels of cytokine production as compare to the other doses tested. Further, mice immunized with rTgPDI demonstrated enhanced survival times and reduced levels of tachyzoites as compared to control mice. Overall, the results from the study demonstrated that immunization with rTgPDI elicited a protective immune response against T. gondii tachyzoites, thus suggesting that this recombinant protein may be a promising candidate for the development of a vaccine to prevent toxoplasmosis.

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Rapid diagnosis of typhoid from blood cultures

Image of Salmonella enterica, courtesy of the CDC

Cara N. Wilder, Ph.D.

Typhoid fever is a life-threatening illness caused by the Gram-negative bacterium, Salmonella enterica subsp. enterica serovar Typhi. This foodborne pathogen is commonly contracted though the consumption of food or water that has been handled by a person shedding S. Typhi, or through the contamination of these products with sewage. Once ingested, the bacteria will inhabit the intestinal tract and bloodstream, resulting in a variety of complications including high fever, stomach pains, septicemia, and death.

Typhoid fever is common throughout most of the developing world, particularly in parts of Asia, Africa, and Latin America. Unfortunately, a number of people within these countries do not have access to a reliable laboratory diagnosis as the appropriate clinical facilities and techniques are not available. Thus, there is an urgent need for an inexpensive, easy-to-use, portable technique that can rapidly and safely diagnose typhoid fever independent of a hospital setting.

In a recent study, Castonguay-Vanier et al. investigated the accuracy and efficacy of a technique combining blood culture amplification of S. Typhi with a S. Typhi antigen rapid diagnostic test (RDT) developed by Standard Diagnostics (Cat. No. 15FK12). When tested against 23 Gram-negative reference pathogens, this assay was able to detect S. Typhi, as well as Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Ndolo. The precision of this assay was further analyzed through the examination of 6,456 blood cultures from 3,028 patients. From this prospective study, the group found that the sensitivity, negative predictive value, specificity, and positive predictive value were 96.7%, 99.5%, 97.9%, and 87.9%, respectively, for patients with proven S. Typhi bacteremia. Overall, these results suggest that the combination of blood culture amplification of S. Typhi with an S. Typhi RDT is promising as an effective, sensitive, and inexpensive tool for the rapid diagnosis of typhoid fever.

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Characterization of Arcobacter species using multi-locus sequence typing

Cara N. Wilder, Ph.D.

Arcobacter is a genus of aerotolerant, Campylobacter-like bacteria first isolated from aborted bovine fetuses by Ellis et al. in 1977. Since then, Arcobacter species have been discovered in a variety of sources, including food, water, animals, and agricultural run-off. In recent years, the prevalence of Arcobacter butzleri and Arcobacter cryaerophilus in food, raw milk, and water has suggested the potential of these organisms to be spread by contaminated consumable products. This is particularly disconcerting as these species are considered to be pathogenic, causing a variety of symptoms such as abdominal pain, nausea, and septicemia.

To aid in the identification, characterization, and epidemiology of Arcobacter strains, Miller et al. developed a multi-locus sequence typing (MLST) scheme based on the analysis of partial, defined sequences from seven Arcobacter housekeeping genes (aspA, atpA(uncA), glnA, gltA, glyA, pgm, and tkt). In this study, a sample set of 374 strains comprising five known Arcobacter species was isolated from a diverse array of sources and geographical regions. The analysis of these strains yielded almost 300 sequence types and 1176 alleles across the seven loci. Overall, this extensive set of genomic sequence data may aid with strain discrimination as well as help track sporadic Arcobacter-related gastroenteritis and potential outbreaks.

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Rapid Detection of Cronobacter spp.

Image of Cronobacter sakazakii, formerly known as
Enterobacter sakazakii. Photo courtesy of CDC and Dr. JJ Farmer.

Cara N. Wilder, Ph.D.

Cronobacter sakazakii is a Gram-negative, foodborne pathogen associated with the use of powdered infant formula. In infants, this bacterium has been found to cause invasive infections with high fatality rates, including neonatal meningitis, sepsis, and necrotizing enterocolitis. Due to the severity of these infections, it is imperative that rapid and sensitive detection assays are available for the microbiological testing of powdered infant formula.

Currently, the routine procedure for the detection of Cronobacter spp. is both arduous and time intensive. To improve upon this, Cai et al., developed a novel molecular-based assay that targets the Cronobacter ompA gene through real-time PCR integrated with high resolution melting (HRM) analysis. This innovative methodology is not only faster than current procedures, reducing detection time from several days to less than 24 hours, it is also probe-free and reduces the risk of PCR carry over. To analyze the specificity and sensitivity of this assay, 11 Cronobacter isolates and 25 reference strains were examined. In this analysis, only Cronobacter spp. produced a positive signal. Moreover, the assay detection limit was found to be 10² CFU/mL, indicating that the assay is highly sensitive. Overall, this established method may provide a rapid, sensitive, and specific molecular tool for the direct detection of Cronobacter spp. in powdered infant formula.

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Identification of Enterococcus species using an automated microarray-based nucleic acid test

Image of Enterococcus sp. Photo courtesy of Janice Haney Carr.

Cara N. Wilder, Ph.D.

Enterococcus faecalis is a Gram-positive commensal bacterium known to inhabit the gastrointestinal tract of humans and animals. In immunologically compromised individuals, E. faecalis is a leading cause of urinary tract infections and nosocomial bacteraemia. This latter condition is of particular concern as it can lead to septic shock or the hematogenous spread of bacteria to other parts of the body, resulting in high rates of mortality and organ failure.

Unfortunately, the treatment of E. faecalis infection is not straightforward as a number of strains exhibit intrinsic and acquired resistance to a variety of antibiotics, including aminoglycosides, cephalosporins, and penicillins. Presently, these drug-resistant strains are commonly treated with vancomycin; however, in some instances the extended use of this antimicrobial drug has resulted in the emergence of vancomycin-resistant strains. To make matters worse, current treatment options for vancomycin-resistant strains is limited. Therefore, the rapid identification of vancomycin-resistant strains is imperative to the successful treatment of infection.

In recent years, advances in diagnostics have yielded a number of sensitive assays for the detection of E. faecalis, including fluorescent in situ hybridization (FISH), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS), and the microarray-based Verigene Gram-Positive Blood Culture test (BC-GP) (Nanosphere). This latter method was recently validated through an examination of 12 Gram-positive targets including E. faecalis, as well as associated drug-resistance determinants. In this study, Buchan et al. used the Verigene BC-GP test to analyze 1,252 positive blood culture broths collected from five clinical centers throughout the United States. The resultant data indicated that the BC-GP test can sensitively and specifically identify E. faecalis and other leading causes of Gram-positive bacteremia directly from positive blood cultures. Further, this assay was capable of detecting the genetic markers vanA and vanB, which confer resistance to vancomycin. Overall, the Verigene BC-GP assay may prE. faecalis, ultimately enhancing public health.
ovide diagnosticians with a more sensitive means of detecting vancomycin-resistant

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