Whole Blood Pathogen Detection for Antibiotic Resistance Genes Identification

Antimicrobial Resistance = Global Public Health Concern

It has been nearly two years since the global healthcare resources have been diverted into the prolonged fight against the SARS-CoV2 pandemic[1], often being forced to put other medical issues and emergencies on hold[2] [3] [4]. Aside from the undeniable threat of the COVID-19 crisis, there are other urgent challenges to the health of the general population, such as the accelerating development of resistance to antimicrobials, seen among many harmful pathogens. Collectively referred to as the antimicrobial resistance (AMR) traits, the development of defense mechanisms to drugs previously effective against the infectious agent, is being increasingly reported in different species and strains of pathogenic bacteria, viruses, fungi, and protozoan parasites. Preventing successful therapy outcomes of treatable infectious diseases in patients, AMR pathogen (superbug) infections have been responsible for more than 700,000 deaths in 2019, with current worst-case scenarios predicting an annual total loss of 10 million lives as of 2050[5] [6] [7].

With the current use of antibiotics alone estimated at 100,000-200,000 tons per year[1], massive and uncontrolled consumption results in a subsequent release of a wide range of substances with antimicrobial properties into the environment. Antibiotics, antivirals, antifungals, antiparasitics, analgesics, and anti-inflammatory drugs are among the most commonly detected classes of pharmaceuticals in the anthropogenic pollution makeup of water, soil, air, and living tissues[2] [3] [4].

The Importance of AMR Genes Identification in Medical Microbiology

As ​​medical microbiology concerns the nature, distribution, and activities of microbes and how they impact health and wellbeing, most particularly as agents of infection[1], it allows for the discovery of effective ways of infectious disease treatments. Besides the illness etiology, the resistance of pathogen and its variants have to be determined to assure susceptibility to drugs of choice for particular infections[2]. Antimicrobial susceptibility is an appropriate test whenever a specimen is collected from a suspected infection site. In the face of active infection, this information, along with the Gram stain and culture, allows selecting an appropriate antimicrobial agent to treat an infection[3].

The latest research indicates AMR is a growing threat i.a. to neonates in low- and middle-income countries, such as India[4]. Gram-negative (GN) Klebsiella spp., Citrobacter spp. and Escherichia spp. with Gram-positive (GP) Staphylococcus spp. are the most commonly identified etiologic agents of bloodstream infections (BSI[5], e.g. Figures 1. and 2.), including in newborns16. Compared with neonates without BSI, all-cause mortality is higher among ones with early and late-onset BSI, with a substantial percentage of AMR strains causing the infections, including GN carbapenem-resistant bacteria, where a critical gap remains in research and development, in particular for antibacterial targeting7 16.

 

Bloodstream infection example. Figure 1. Colorized SEM of methicillin-resistant Staphylococcus aureus (MRSA) bacteria on the surface of a wound dressing[1]. Figure 2. The hypothesized life cycle of methicillin-resistant Staphylococcus aureus isolates that cause persistent bacteremia in the context of endovascular infection[2]. GP bacteria acquire resistance to beta-lactam antibiotics through the production of a protein called PBP2a, which is able to avoid the inhibitory effects of the antibiotics. This is the mechanism by which MRSA is able to persist despite treatment with multiple beta-lactam antibiotics[3].

Antibiotic resistance is thus not a future risk, but one of another epidemic threats that, without appropriate action could, like COVID-19, turn into a pandemic costing humanity millions of lives and dollars. We can no longer “sit tight and assess”[1] the situation, we must be proactive to get off course towards a post-antibiotic era, for which the invention of rapid diagnostic methods is essential[2] [3] [4]. The importance of AMR Genes Identification (ID) is evidenced by the Global Antimicrobial Resistance and Use Surveillance System – GLASS – which has been conceived by the WHO to incorporate data from infections of AMR in humans, the use of antimicrobial medicines, AMR in the food chain and in the environment, filling knowledge gaps and to inform strategies at all levels7, helping in the development of more effective therapies such us the clinical use of bacteriophages and new drugs[5].

State of the Art Pathogen Detection Methods: Possibilities & Limitations

As routine microbiological screening includes resistant variants identification, the potential patient diagnosis waiting times should be as short as possible, especially for serious infections such as sepsis. However, in light of the overall AMR threat, not only the character of the resistance should be taken into account during pathogen ID in medical practice, but also the molecular mechanisms determining the occurrence and evolution of antimicrobial traits through genome sequencing13 [1]. Therefore, a need for fast but highly sensitive and efficient methods emerges while looking for improvements to current State of the Art Pathogen ID Methods14 [2].

  • Conventional Blood Cultures

Traditional phenotypic ID methods of bacteria and fungi from blood cultures (i.e. inoculation of a patient’s blood sample on special microbiological media) and determining antimicrobial susceptibility (i.e. growing isolated bacteria on media with certain antimicrobials) last at least 24 hours due to their requirement for microbial growth rates, delaying optimal therapy and negatively impacting patient outcomes. In general, the detection and identification is a process taking two days for most organisms or even longer for fastidious organisms. Moreover, blood cultures do not provide the information about the nature of the resistance e.g. specific genes or plasmids determining observed culture resistant growth, however in some molecular research methods the amount of input pathogen material for testing is relatively high, so that the growth of bacterial cultures must also be taken into account13 14 19 26 [1] [2].

  • Immunological Assays

Immunological Assays look at the pathogen components (antigens) by antibodies raised against that pathogen specific antigens. Current immuno-methods include antigen binding, latex or coagglutination products for specific identification, Lateral Flow Immunoassays with phages and direct biochemical tests, providing more rapid ID of bacteria or fungi, and in some cases antimicrobial resistance markers, from positive blood cultures, as well as directly from whole blood. Sensitivities, specificities, and predictive values for the ​​Streptococcaceae products have generally been high, but for Staphylococcaceae, the sensitivities in particular have been low. Immunological testing can be also applied directly towards specific nucleic acids, providing an opportunity for more specific strain ID14 32.

  • Nucleic Acid Detection Schemes

To investigate this multitude of mechanisms without relying on phenotypic observations, there is a large repertoire of molecular diagnostics technologies already made their way to clinical routine, as e.g. polymerase chain reaction (PCR) and real time (RT) PCR-based detection or whole genome sequencing (WGS). The currently most popular used tests are based on RT-PCRs due to their relatively good sensitivity, specificity and speed. Using highly conserved ribosomal RNA (rRNA) genes as targets usually allows a higher sensitivity since multiple copies of this genes are present in the genome. The application as well as the variability of PCR are multitudinous. The most important in clinical diagnostics are the conventional and the real-time PCR, which allows to observe the amplification live using unspecifically intercalating dyes or specific DNA seq which give rise to a fluorescence signal only after hybridizing to the amplicon. However, these tests can only identify a low number of targets because of the limited availability of differentiable fluorescence dyes. In addition, with an increasing degree of multiplexing, the sensitivity and specificity of PCRs is reduced due to unintended amplification products and primer dimer formation26 28 [3] [4].

Strategies for Improvement of AMR ID

Both phenotypic and molecular-based (PCR-derived) methods are considered Conventional AMR Diagnostic Techniques, with Immunological Assays seemingly fitting as Microfluidic Technologies. Genome Sequencing and Metagenomics are Non-Conventional Methods. These newly introduced second and third generation sequencing approaches have paved the way for single genome sequencing, as well as for the characterization of complex microbial communities and the identification of antibiotic resistance determinants. Whole metagenome sequencing (WMS) and analysis of genetic material in patient samples allows for the identification of AMR genes directly from clinical specimens without the need for prior isolation or identification of specific pathogen[1].

mNGS technologies (metagenomic Next Generation Sequencing) have become an essential tool for unbiased, culture-independent diagnosis as well as drug and diagnostic tests development by enabling the rapid identification and surveillance of resistance mechanisms. Including a complete genomic sequence provided by the metagenomics advancements represent the highest practicable level of structural detail on the individuating traits of an organism or population. It can be used to provide more reliable microbial identification, definitive phylogenetic relationships, and a comprehensive catalog of traits relevant for epidemiological investigations. This is having a major impact on outbreak investigations and the diagnosis and treatment of infectious diseases, as well as the practice of microbiology and epidemiology[2] [3] [4]. However, the major bottleneck keeping mNGS from mass adoption in clinical microbiology, is human DNA contamination with patient’s genetic information depletion being the main cost in rapid pathogen ID and a timely treatment35 [5] [6] .

Devin® is a novel blood fractionation filter device that can be applied to efficiently deplete human cellular DNA background and enrich microorganisms in the whole blood samples in less than 5 minutes37. With ten most frequent microorganisms causing bacteremia and fungemia in adults belonging to the Enterobacteriaceae, Staphylococcaceae and ​​Streptococcaceae, families14, whose AMR strains are listed by the CDC as either concerning, serious or urgent threats[7], the PaRTI-Seq® test, a metagenomic sequencing workflow built upon Devin® can identify these potential pathogens from whole blood samples within 24 hours with a sensitivity of 102 genome copies per milliliter. Increasing the scope of available genomic screening for the presence of antimicrobial resistance factors using PaRTI-Seq® and Devin® technology could not only speed up the diagnostic process, especially during BSI but also improve the efficiency of the AMR screening process, increasing existing antimicrobial databases with crucial data sharing and exchange4 37. Devin® and PaRTI-Seq® have the potential for saving the sequencing cost significantly and constitute a faster method for AMR ID, especially from whole blood samples37.

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