The Rise of Multidrug-Resistant Strains and Need for New Therapeutic Approaches

Cara N. Wilder, Ph.D.

The discovery of antibiotics in the early twentieth century has revolutionized the treatment of infectious diseases, saving millions of lives and easing the suffering of many. However, as the structure and function of antibiotics has evolved through the efforts of biotech and pharmaceutical companies, microorganisms have evolved in parallel, fashioning novel and effective methods to avoid therapeutic biocide. In the last several decades, this concern has become more pronounced with the emergence of multidrug-resistant organisms in both community- and hospital-acquired infections, resulting in increased morbidity, mortality, and health-care expense. Here, we will discuss the growing threat of antimicrobial resistance, novel therapeutic approaches, and the importance of antimicrobial-resistant reference strains in reducing the emergence and spread of multidrug-resistant infections.

Antimicrobial resistance is currently recognized as one of the greatest threats to human health worldwide¹. According to the World Health Organization (WHO), antimicrobial-resistant strains are present in all parts of the world, and new resistance mechanisms continue to emerge and spread globally². In the United States alone, at least 2 million people become infected with antibiotic-resistant bacteria, with approximately 23,000 associated deaths each year³. These patients are typically at an increased risk of more debilitating clinical outcomes and death, and consume more healthcare resources as compared to patients infected with a drug-sensitive strain of the same species². Infection with these microorganisms is further complicated by the limited number of effective therapeutic options. In some cases, clinicians have resorted to using older drugs, for which there may be limited data on safety and efficacy to guide the dosage or duration^4,5. Overall, this highlights the dire need for novel and effective therapeutics to treat multidrug-resistant infections.

As the number of antibiotics effective against multidrug-resistant strains is beginning to dwindle, there have been a variety of efforts made to evaluate novel therapeutic options. For example, one novel mechanism used in a 2010 study by Nicolosi et al. employed the use of fusogenic liposomes to transport vancomycin into Gram-negative cells⁶. Under normal conditions, vancomycin is only used to treat Gram-positive organisms. It is inactive against Gram-negative bacteria due to the different mechanism in which Gram-negative bacteria produce their cell walls, as well as other factors relating to entering the outer membrane of Gram-negative organisms. Fusogenic liposomes offer an alternative method for the delivery of the antibiotic across the gram-negative outer membrane. These small unilamellar liposome vesicles are able to adhere to and fuse with the external membrane; thus, if you load these liposome vesicles with an antibiotic such as vancomycin, you can transport the antibiotic across the membrane. Overall, this offers a promising mechanism for extending the use of current antibiotics.

Another emerging therapeutic approach against multidrug-resistant strains is the use of antimicrobial peptides. For example, in a 2010 study by Routsias et al., the group found that human beta-defensin 2, which is a naturally occurring peptide, exhibited high bactericidal activity against Acinetobacter baumannii. As antimicrobial peptides are naturally occurring, are bactericidal, and are broad-spectrum, they offer a promising mechanism for treating drug-resistant infections⁷. Antisense agents also provide another potential therapeutic approach for treating drug-resistant infections by inhibiting resistance mechanisms directly at the nucleic acid level. These oligonucleotides could potentially be designed to bind specific mRNA or DNA sequences that confer resistance to antibiotics, thus blocking either translation or gene transcription, rendering the cell susceptible to antibiotic treatment. However, as these nucleic acids would have no intrinsic antibacterial activity of their own, they would have to be used in conjunction with antibiotics as needed⁸.

Bacteriophage T4. Photo credit: David
Gregory and Debbie Marshall

Before the use of antibiotics, some efforts were made to use bacteriophages for the treatment of bacterial infections. Following the first isolation of a bacteriophage in 1917, bacteriophages have been extensively used and developed in several countries throughout the former Soviet Union⁹. The primary advantage to this form of treatment is the specificity of a bacteriophage toward a specific bacterial target. This would allow for direct killing of a bacterial pathogen without harming the resident microflora or the patient. In turn, this would help reduce the likelihood of opportunistic, secondary infections from occurring. However, this specificity also poses a potential problem in that to effectively treat an infection, the causative agent must be properly diagnosed^10,11. Further, it is possible for bacterial strains to become resistant to certain types of bacteriophages following continual exposure; however, this could be potentially overcome through the use of genetically modified bacteriophages.

Lastly, there are some efforts being made to help support the development of new antibiotics. In 2010, the Infectious Diseases Society of America (IDSA) launched a collaboration entitled the “10 x ‘20” initiative to help create a global antibacterial drug research and development enterprise with the power to develop 10 new, safe, and effective antibiotics by the year 2020¹². Here, the IDSA is supporting the development of 10 new systemic antibacterial drugs through the development of new drugs from existing antibiotic classes or through the discovery of novel antibiotic classes not previously known¹². To date, the initiative has spurred the development of two novel antibiotics, which were recently approved in the United States by the Food and Drug Administration (FDA)^13,14.

Regardless of the type of therapeutic approach, it is important to ensure that the novel treatment is both safe for patient use and effective against a variety of drug-resistant strains. This can be evaluated through in vitro analyses using authenticated strains and cell lines. Characterized microbial strains with known antibiotic susceptibility profiles are ideal for evaluating the efficacy of a novel therapeutic against different species that vary in their resistance/susceptibility profiles. These strains would also be effective in the development and validation of novel detection methods as it would allow a mechanism to evaluate the sensitivity and specificity against various species and mechanisms of drug resistance. For analyzing the safety of the therapeutic, primary cells derived from various organ systems and donors can provide a quick mechanism for screening therapeutics for any potential toxic effects.

To support this need, ATCC offers a complete set of solutions to advance multidrug-resistance research, including drug-resistant microbial reference strains from various clinical and environmental sources, primary cells for drug toxicity screening studies, as well as media and reagents that support growth. Each of these reference materials are produced under ISO 9001:2008 certified and ISO/IEC 17025:2005 accredited processes, ensuring reliability and reproducibility of research data. Further, to ensure that each of these products are of the highest quality before they reach your laboratory, ATCC has authenticated each strain using phenotypic and genotypic analyses to guarantee species identification, culture purity, and biochemical consistency.

Overall, multidrug-resistant microbial strains are continually implicated in a number of infections worldwide, resulting in significant increases in morbidity, mortality, and health care expense. To help control the emergence and spread of these pathogens, discovering new and effective treatment methods is of the upmost importance. To support this need, ATCC offers fully authenticated, characterized strains and cell lines for use as positive controls. These cultures are ideal for evaluating the efficacy and toxicity of novel therapeutic treatments. In conclusion, microorganisms are rapidly evolving or acquiring mechanisms of resistance to antibiotics. Only through aggressive action and scientific ingenuity can the emergence and spread of multidrug resistance be prevented.


References

1. Walker, B. et al. Environment. Looming global-scale failures and missing institutions. Science 325, 1345-1346, doi:10.1126/science.1175325 (2009).
2. WHO. Fact sheet N°194 - Antimicrobial resistance, <http://www.who.int/mediacentre/factsheets/fs194/en/> (2014).
3. CDC. Antibiotic/Antimicrobial Resistance, <http://www.cdc.gov/drugresistance/> (2014).
4. Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 48, 1-12, doi:10.1086/595011 (2009).
5. Cassir, N., Rolain, J. M. & Brouqui, P. A new strategy to fight antimicrobial resistance: the revival of old antibiotics. Frontiers in microbiology 5, 551, doi:10.3389/fmicb.2014.00551 (2014).
6. Nicolosi, D., Scalia, M., Nicolosi, V. M. & Pignatello, R. Encapsulation in fusogenic liposomes broadens the spectrum of action of vancomycin against Gram-negative bacteria. International journal of antimicrobial agents 35, 553-558, doi:10.1016/j.ijantimicag.2010.01.015 (2010).
7. Routsias, J. G., Karagounis, P., Parvulesku, G., Legakis, N. J. & Tsakris, A. In vitro bactericidal activity of human beta-defensin 2 against nosocomial strains. Peptides 31, 1654-1660, doi:10.1016/j.peptides.2010.06.010 (2010).
8. Woodford, N., Wareham, D. W. & Group, U. K. A. A. S. Tackling antibiotic resistance: a dose of common antisense? The Journal of antimicrobial chemotherapy 63, 225-229, doi:10.1093/jac/dkn467 (2009).
9. Mandal, S. M. et al. Challenges and future prospects of antibiotic therapy: from peptides to phages utilization. Frontiers in pharmacology 5, 105, doi:10.3389/fphar.2014.00105 (2014).
10. Bragg, R., van der Westhuizen, W., Lee, J. Y., Coetsee, E. & Boucher, C. Bacteriophages as potential treatment option for antibiotic resistant bacteria. Advances in experimental medicine and biology 807, 97-110, doi:10.1007/978-81-322-1777-0_7 (2014).
11. Tiwari, R., Dhama, K., Kumar, A., Rahal, A. & Kapoor, S. Bacteriophage therapy for safeguarding animal and human health: a review. Pakistan journal of biological sciences : PJBS 17, 301-315 (2014).
12. ISDA. The 10 × '20 Initiative: Pursuing a Global Commitment to Develop 10 New Antibacterial Drugs by 2020. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 50, 1081-1083 (2010).
13. Boucher, H. W. et al. 10 x '20 Progress--development of new drugs active against gram-negative bacilli: an update from the Infectious Diseases Society of America. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 56, 1685-1694, doi:10.1093/cid/cit152 (2013).
14. Murray, B. Progress in 10 x '20 Initiative Shows Promise but More Action Needed. Infection Control Today (June 23, 2014).

The role of microorganisms in the induction of cancer

Cara N. Wilder, Ph.D.

Since the beginning of the 20^th century, microorganisms have been known to play a significant role in the development of cancer in animals¹. However, our understanding on the extent of this issue in humans is still continuing to expand. Currently, it is estimated that microbial infections are linked to approximately 15-20% of human cancers throughout the world^1-3. This presents an enormous potential for the prevention of cancer through the control of infectious disease.

In a 2006 International Journal of Cancer study, it was estimated that the total number of cancers caused by microbial infection in 2002 was approximately 17.8% of the global cancer burden². Of this subset, most of the burden was attributable to viral infections (12.1%), followed by infection with Helicobacter pylori (5.6%), with a small proportion due to parasitic infection (0.1%) (Table 1)². Further, the percentage of these infection-attributable cancers was higher in developing countries as compared to developed countries, reflecting the higher prevalence of these organisms, and perhaps the need for more readily-available healthcare.

Table 1. Examples of microbial-associated cancers^2,4

Agent	Type	Cancer
Epstein-Barr virus (EBV)	Herpesvirus	Hodgkin’s lymphoma, Burkitt’s lymphoma, Nasopharyngeal carcinoma
Helicobacter pylori	Bacterium	Gastric adenocarcinoma
Hepatitis B virus (HBV)	Hepadnavirus	Hepatocellular carcinoma
Hepatitis C virus (HCV)	Flavivirus	Hepatocellular carcinoma
Human herpesvirus 8 (HHV-8)	Herpesvirus	Kaposi’s sarcoma
Human papillomavirus (HPV)	Papillomavirus	Anogenital carcinoma, Oropharyngeal carcinoma
Human T-lymphotropic virus 1 (HTLV-1)	Retrovirus	Adult T-cell lymphoma
Opisthorchis viverrini	Trematode	Cholangiocarcinoma
Schistosoma haematobium	Trematode	Bladder squamous cell carcinoma

The ability of these microorganisms to cause cancer greatly depends on the context of the host-microbial relationship. These interactions can vary significantly due to a number of factors such as the oncogenic potential of the strain, how the strain interacts with the host’s cells and immune system, if the infection causes long-term inflammation, host genotype and phenotype, environmental factors, microbial load, and the composition of the host’s microbiome^1,4. Further, there is some speculation that the ability for certain cancers to run within a family may in part be due to the intra-familial transmission of oncogenic microorganisms. In this latter case, indigenous organisms acquired from a family member may be preadapted to the next host, which could modify the risk of disease^5,6. Regardless of the mechanism, it is clear that preventing or controlling microbial infections may help reduce the number of cancer cases throughout the world.

There are a number of methods that can be used to help prevent cancers associated with microbial infection. Taking measures to avoid exposure to strains associated with infection-attributable cancers, such as circumventing the habitats of harmful species or abstaining from activities that promote transmission, can help reduce the likelihood of infection. Another approach is through the use of vaccination. Currently, vaccines are available against the hepatitis B virus as well as human papillomavirus types 16 and 18. As these vaccines have already demonstrated efficacy in preventing infection, they have the potential to significantly reduce healthcare cost as well as the incidence of liver and anogenital cancer, respectively. If vaccination is not possible, available antimicrobials can be used to help mediate or cure an established infection. For example, in the treatment of H. pylori, the administration of triple therapy regimens for 10-14 days consisting of a proton pump inhibitor, amoxicillin, and clarithromycin are typically recommended, and have reported cure rates from 85-90%⁷.

Overall, infection-attributable cancers represent a significant, albeit preventable, proportion of the global cancer burden. There are currently a number of therapeutic options to help stave off, control, or treat many known oncogenic infections; however, more research is still needed to expand the range of effective therapeutic options as well as help identify additional microbial strains that have the potential to cause cancer. To that end, a number of reputable biological resource centers, such as ATCC, offer a variety of microorganisms, nucleic acids, and cell lines that can be used to help support the research in this field. Only through hard work and dedicated research can we help significantly reduce the number of cancer cases throughout the world.

References

1. American Cancer Society. Infections that can lead to cancer, <http://www.cancer.org/cancer/cancercauses/othercarcinogens/infectiousagents/infectiousagentsandcancer/infectious-agents-and-cancer-intro> (2014).
2. Parkin, D. M. The global health burden of infection-associated cancers in the year 2002. International journal of cancer. Journal international du cancer 118, 3030-3044, doi:10.1002/ijc.21731 (2006).
3. WHO. Global status report on non communicable disease 2010. WHO Library Cataloguing-in-Publication Data (2011).
4. Blaser, M. J. Understanding Microbe-Induced Cancers. Cancer Prevention Research 1, 15-20 (2008).
5. Blaser, M. J. & Kirschner, D. The equilibria that allow bacterial persistence in human hosts. Nature 449, 843-849, doi:10.1038/nature06198 (2007).
6. Blaser, M. J., Nomura, A., Lee, J., Stemmerman, G. N. & Perez-Perez, G. I. Early-life family structure and microbially induced cancer risk. PLoS medicine 4, e7, doi:10.1371/journal.pmed.0040007 (2007).
7. Santacroce, L. & Bhutani, M. Helicobacter Pylori Infection Treatment & Management, <http://emedicine.medscape.com/article/176938-treatment> (2014).

Using Next-Generation Sequencing for Infectious Disease Research – Understanding Microbial Pathogens from Within

Cara N. Wilder, Ph.D.

Ever since James Watson and Francis Crick derived the double-helix model for the structure of DNA in 1953, the field of genomics has exponentially burgeoned into a vast array of molecular technologies. Of the current assortment of available genomic tools, next-generation sequencing (NGS) has proven to be of great value, particularly in the field of infectious disease. Here, the knowledge of DNA and RNA sequences has become indispensable for a variety of applications, including microbial identification and detection, evaluating the evolution and regulation of virulence, identifying drug resistance markers, detecting antigenic targets for vaccine development, and delineating community outbreaks.

NGS technologies are typically described as non-Sanger-based high-throughput DNA sequencing platforms that enable the analysis of millions of DNA fragments in parallel¹. This ability to multiplex provides unprecedented scalability and has contributed to a gradual reduction in reagent costs, allowing whole-genome sequencing (WGS) to become accessible and practical to researchers around the world. Over the last decade, a number of platforms and methods have been developed that enable de novo sequencing, re-sequencing, transcriptome profiling, and cDNA sequencing.

In the field of infectious disease, NGS affords a means for reviewing the complete genetic make-up of microbial pathogens through de novo sequencing or re-sequencing, regardless of the fastidious or non-fastidious nature of the strain. This ability to review an entire genome, or individual genes or operons, provides a unique opportunity for clinical diagnostics, the identification of potential antigenic targets for vaccine development, and epidemiological surveillance. Currently, the diagnosis of microbial infections typically begins with patient observation and the collection of specimens for microbiological processing for various diagnostic tests. Depending on the causative agent, these diagnostics tests may include serology and analyzing specimens for the presence of immune cells via microscopy. For some bacterial infections, culture-based growth techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and polymerase chain reaction (PCR) are also used for microbial identification and analysis. If employed, NGS could potentially be used to identify a pathogen without prior knowledge of the target, as well as discern the presence of any virulence determinants or genes conferring drug resistance^2-4. In turn, this would provide an additional clinical tool that may help patients receive a more precise diagnosis and efficient treatment regimen.

In addition to clinical diagnostics, NGS offers a potential tool for vaccine development and evaluation. For example, NGS can allow for the surveillance of pathogen evolution. For rapidly mutating pathogens such as the influenza virus, this could provide an opportunity to help identify antigenic drift within the microbial population and use that knowledge to create a better, more specific vaccine. NGS could also be used for reverse vaccinology, where pathogen sequences are used to identify putative surface antigens that could potentially be targeted for the development of a novel vaccine preparation⁴. In accordance with this latter application, the expression of the putative surface antigens can also be examined through the evaluation of RNA sequences during different stages throughout the pathogen life cycle. As some strains require more than one species for survival and replication, analyzing RNA expression can help ensure that the vaccine target is actively expressed in the human host to ensure an efficient immunological response. Lastly, NGS could be applied in vaccine safety analyses by detecting the presence of viral contaminants in cell culture used in vaccine development or identifying virulent mutations in live-attenuated vaccines prepared with highly pathogenic, genetically unstable strains prior to the use of the vaccine^5,6. Overall, NGS could help contribute to the development of safer vaccines that provide a more effective immune response.

Lastly, NGS is gradually being employed in the epidemiological analysis of cultures for outbreak detection and infection control. Using genomics, outbreak strains have been identified at the species level and below with extraordinarily high resolution. Through the analysis of these strains, population genetic studies can contribute to a better understanding of important genetic markers among strains while also tracking transmission events during outbreaks. In turn, this allows for the development of a tailored control program to help limit the further spread of disease⁷. A recent example of the use of NGS in epidemiological surveillance was during the analysis of a 2011 hospital outbreak of methicillin-resistant Staphylococcus aureus (MRSA)⁸. Here, WGS was used to identify hospitalized infant patients infected with MRSA and determine the source of transmission. Through this study, 26 related cases of MRSA were identified and it was demonstrated that transmission occurred within the hospital between mother and patient, among mothers on a postnatal ward, and in the community. Moreover, WGS confirmed that MRSA carriage by a staff member allowed the outbreak to persist during periods of no known infection. Overall, this study demonstrated that NGS technology enabled a more precise identification of patients involved in an outbreak, allowing for the implementation of an effective infection control program that helped halt the spread of disease.

Despite the numerous advantages of NGS in the analysis of infectious diseases, there are a few challenges that may need to be overcome. For example, for NGS data to be reliable, particularly with regard to re-sequencing and microbial identification, it is important that validated sequence data is readily accessible. Well-renowned sequence databases (e.g. GenBank) provide an annotated collection of publically available genome sequences from authenticated type strains, such as those obtained from prominent biological resource centers like ATCC. As type strains are the nomenclatural origin of a species or subspecies, they are typically fully authenticated and exhibit all of the relevant phenotypic and genotypic properties described in original taxonomic circumscriptions, making them ideal reference standards for NGS. Extensive bioinformatics to analyze sequences from assembly to annotation, which necessitates expertise and time, could pose another challenge for NGS clinical use. This can be potentially circumvented through the development of automated tools for sequence analysis combined with the assemblage of exhaustive databases containing all known genome sequences³. Lastly, there is the possibility that host DNA will be analyzed alongside microbial nucleic acids, which may make it difficult to discern the target genomics. To overcome this, the microbial to host nucleic acid ratio must be increased through procedures such as hydrolysis or chemical treatment⁴.

In conclusion, clinical microbiology laboratories face a number of challenges regarding the diagnosis and effective treatment of infectious diseases. NGS could provide an additional insight to aid in clinical diagnostics while offering an effective mechanism for the identification of potential vaccine targets and a way to track outbreaks. Only through continual efforts in the advancement of this technology can NGS become fully ingrained in the field of infectious disease as a routine diagnostic tool.

References

1. Nature. Next-generation sequencing – Definition. http://www.nature.com/subjects/next-generation-sequencing, 2015. 
2. Wain J, Mavrogiorgou E. Next-generation sequencing in clinical microbiology. Expert Rev Mol Diagn 13(3): 225-227, 2013. 
3. Fournier PE, et al. Modern clinical microbiology: new challenges and solutions. Nat Rev Microbiol 11: 574-585, 2013. 
4. Lecuit M, Eloit M. The diagnosis of infectious disease by whole genome next generation sequence: a new era is opening. Front Cell Infect Microbiol 4: 25, 2014. 
5. Luciani F, et al. Next generation deep sequencing and vaccine design: today and tomorrow. Trends in Biotechnology 30(9): 443-452, 2012. 
6. Neverov A, Chumakov K. Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines. Proc Natl Acad Sci 107(46): 20063-20068, 2010. 
7. Tang P, Gardy JL. Stopping outbreaks with real-time genomic epidemiology. Genome Med 6(11): 104, 2014. 
8. Harris SR, et al. Whole-genome sequencing for analysis of an outbreak of methicillin-resistant Staphylococcus aureus: a descriptive study. Lancet Infect Dis 13(2): 130-136, 2013.