Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

mRNA vaccine platforms to prevent bacterial infections.

Bacterial infections are an urgent public health priority. The application of mRNA vaccine technology to prevent bacterial infections is a promising therapeutic strategy undergoing active development. This article discusses recent advances and limitations of mRNA vaccines to prevent bacterial diseases and provides perspectives on future research directions.

Inferring Pathways of Oxidative Folding from Prefolding Free Energy Landscapes of Disulfide-Rich Toxins.

Short, cysteine-rich peptides can exist in stable or metastable structural ensembles due to the number of possible patterns of formation of their disulfide bonds. One interesting subset of this peptide group is the conotoxins, which are produced by aquatic snails in the family Conidae. The µ conotoxins, which are antagonists and blockers of the voltage-gated sodium channel, exist in a folding spectrum: on one end of the spectrum are more hirudin-like folders, which form disulfide bonds and then reshuffle them, leading to an ensemble of kinetically trapped isomers, and on the other end are more BPTI-like folders, which form the native disulfide bonds one by one in a particular order, leading to a preponderance of conformations existing in a single stable state. In this Article, we employ the composite diffusion map approach to study the unified free energy surface of prefolding µ-conotoxin equilibrium. We identify the two most important nonlinear collective modes of the unified folding landscape and demonstrate that in the absence of their disulfides, the conotoxins can be thought of as largely disordered polymers. A small increase in the number of hydrophobic residues in the protein shifts the free energy landscape toward hydrophobically collapsed coil conformations responsible for cysteine proximity in hirudin-like folders, compared to semiextended coil conformations with more distal cysteines in BPTI-like folders. Overall, this work sheds important light on the folding processes and free energy landscapes of cysteine-rich peptides and demonstrates the extent to which sequence and length contribute to these landscapes.

Lipoprotein capture ELISA method for the sensitive detection of amphiphilic biomarkers.

Enzyme-linked immunosorbent assays (ELISAs) are widely employed for the detection of protein targets due to their ease of use, sensitivity, and potential for high-throughput analyses. However, the use of ELISAs to detect non-protein targets such as lipids and amphiphiles is complicated by the physical properties of these molecules, which affects their association with functional surfaces and recognition ligands. Here, we developed a unique lipoprotein capture ELISA in which the natural association between lipoproteins and amphiphilic molecules facilitates detection of the target biomarker in a physiologically relevant conformation. An assay to detect the glycolipid lipoarabinomannan (LAM), a cell membrane component and virulence factor associated with Mycobacterial infections, was developed as a proof of concept.

Conditions for Handling and Optimal Storage of Mycolactone.

The successful isolation of mycolactone in a laboratory or from a clinical sample relies on proper handling and storage of the toxin. Mycolactone is a light-sensitive and an amphiphilic toxin produced by Mycobacterium ulcerans. The biochemistry of the toxin makes it unstable in aqueous matrices such as blood, which causes it to self-aggregate or present in complex with carrier molecules. This biochemistry also impacts the use of the toxin in vitro, in that it tends to aggregate and stick to substrates in an aqueous environment, which alters its physiological presentation and limits its availability in a sample. Glass materials (i.e., tubes, vials, syringes, plates) should be used when possible to avoid loss of mycolactone sticking to plastic surfaces. Dark containers such as amber vials or aluminum-foil wrapped tubes should be used to avoid photodegradation of the toxin upon exposure to light. Sample storage in organic solvents is ideal for mycolactone stability and recovery; however, this is not always amenable as multiple diagnostic assays might be performed on a single sample (such as PCR or ELISA). In these cases, samples can be stored in an aqueous solution containing a small amount of detergent to enhance recovery of the toxin, and in order to avoid aggregation. Therefore, the downstream manipulations should be carefully considered prior to sample collection and storage. Here we present considerations for the optimal handling and storage of mycolactone in order to obtain quality yield of the toxin for various research and diagnostic applications.

Fast Evaluation of Viral Emerging Risks (FEVER): A computational tool for biosurveillance, diagnostics, and mutation typing of emerging viral pathogens.

Viral pathogens can rapidly evolve, adapt to novel hosts, and evade human immunity. The early detection of emerging viral pathogens through biosurveillance coupled with rapid and accurate diagnostics are required to mitigate global pandemics. However, RNA viruses can mutate rapidly, hampering biosurveillance and diagnostic efforts. Here, we present a novel computational approach called FEVER (Fast Evaluation of Viral Emerging Risks) to design assays that simultaneously accomplish: 1) broad-coverage biosurveillance of an entire group of viruses, 2) accurate diagnosis of an outbreak strain, and 3) mutation typing to detect variants of public health importance. We demonstrate the application of FEVER to generate assays to simultaneously 1) detect sarbecoviruses for biosurveillance; 2) diagnose infections specifically caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and 3) perform rapid mutation typing of the D614G SARS-CoV-2 spike variant associated with increased pathogen transmissibility. These FEVER assays had a high in silico recall (predicted positive) up to 99.7% of 525,708 SARS-CoV-2 sequences analyzed and displayed sensitivities and specificities as high as 92.4% and 100% respectively when validated in 100 clinical samples. The D614G SARS-CoV-2 spike mutation PCR test was able to identify the single nucleotide identity at position 23,403 in the viral genome of 96.6% SARS-CoV-2 positive samples without the need for sequencing. This study demonstrates the utility of FEVER to design assays for biosurveillance, diagnostics, and mutation typing to rapidly detect, track, and mitigate future outbreaks and pandemics caused by emerging viruses.

Optical Biosensor Platforms Display Varying Sensitivity for the Direct Detection of Influenza RNA.

Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention's (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC's probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.

Macrolides: From Toxins to Therapeutics.

Macrolides are a diverse class of hydrophobic compounds characterized by a macrocyclic lactone ring and distinguished by variable side chains/groups. Some of the most well characterized macrolides are toxins produced by marine bacteria, sea sponges, and other species. Many marine macrolide toxins act as biomimetic molecules to natural actin-binding proteins, affecting actin polymerization, while other toxins act on different cytoskeletal components. The disruption of natural cytoskeletal processes affects cell motility and cytokinesis, and can result in cellular death. While many macrolides are toxic in nature, others have been shown to display therapeutic properties. Indeed, some of the most well known antibiotic compounds, including erythromycin, are macrolides. In addition to antibiotic properties, macrolides have been shown to display antiviral, antiparasitic, antifungal, and immunosuppressive actions. Here, we review each functional class of macrolides for their common structures, mechanisms of action, pharmacology, and human cellular targets.

Nucleic Acid-Based Sensing Techniques for Diagnostics and Surveillance of Influenza.

Influenza virus poses a threat to global health by causing seasonal outbreaks as well as three pandemics in the 20th century. In humans, disease is primarily caused by influenza A and B viruses, while influenza C virus causes mild disease mostly in children. Influenza D is an emerging virus found in cattle and pigs. To mitigate the morbidity and mortality associated with influenza, rapid and accurate diagnostic tests need to be deployed. However, the high genetic diversity displayed by influenza viruses presents a challenge to the development of a robust diagnostic test. Nucleic acid-based tests are more accurate than rapid antigen tests for influenza and are therefore better candidates to be used in both diagnostic and surveillance applications. Here, we review various nucleic acid-based techniques that have been applied towards the detection of influenza viruses in order to evaluate their utility as both diagnostic and surveillance tools. We discuss both traditional as well as novel methods to detect influenza viruses by covering techniques that require nucleic acid amplification or direct detection of viral RNA as well as comparing advantages and limitations for each method. There has been substantial progress in the development of nucleic acid-based sensing techniques for the detection of influenza virus. However, there is still an urgent need for a rapid and reliable influenza diagnostic test that can be used at point-of-care in order to enhance responsiveness to both seasonal and pandemic influenza outbreaks.

A centrifugal microfluidic cross-flow filtration platform to separate serum from whole blood for the detection of amphiphilic biomarkers.

The separation of biomarkers from blood is straightforward in most molecular biology laboratories. However, separation in resource-limited settings, allowing for the successful removal of biomarkers for diagnostic applications, is not always possible. The situation is further complicated by the need to separate hydrophobic signatures such as lipids from blood. Herein, we present a microfluidic device capable of centrifugal separation of serum from blood at the point of need with a system that is compatible with biomarkers that are both hydrophilic and hydrophobic. The cross-flow filtration device separates serum from blood as efficiently as traditional methods and retains amphiphilic biomarkers in serum for detection.

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya.

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003-2010. Nine isolates were identified as S. Typhimurium sequence type 313 while the other two were S. Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S. Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

Formulation of stabilizer-free, nontoxic PLGA and elastin-PLGA nanoparticle delivery systems.

Biocompatible nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) are used as drug and vaccine delivery systems because of their tunability in size and sustained release of cargo molecules. While the use of toxic stabilizers such as polyvinyl alcohol (PVA) limit the utility of PLGA, stabilizer-free PLGA nanoparticles are rarely used because they can be challenging to prepare. Here, we developed a tunable, stabilizer-free PLGA nanoparticle formulation capable of encapsulating plasmid DNA and demonstrated the formation of an elastin-like polymer PLGA hybrid nanoparticle with exceptional stability and biocompatibility. A suite of PLGAs were fabricated using solvent evaporation methods and assessed for particle size and stability in water. We find that under physiological conditions (PBS at 37˚C), the most stable PLGA formulation (P4) was found to contain a greater L:G ratio (65:35), lower MW, and carboxyl terminus. Subsequent experiments determined P4 nanoparticles were as stable as those made with PVA, yet significantly less cytotoxic. Variation in particle size was achieved through altering PLGA stoichiometry while maintaining the ability to encapsulate DNA and were modified with elastin-like polymers for increased immune tolerance. Overall, a useful method for tunable, stabilizer-free PLGA nanoparticle formulation was developed for use in drug and vaccine delivery, and immune targeting.

Vaccine Advances against Venezuelan, Eastern, and Western Equine Encephalitis Viruses.

Vaccinations are a crucial intervention in combating infectious diseases. The three neurotropic Alphaviruses, Eastern (EEEV), Venezuelan (VEEV), and Western (WEEV) equine encephalitis viruses, are pathogens of interest for animal health, public health, and biological defense. In both equines and humans, these viruses can cause febrile illness that may progress to encephalitis. Currently, there are no licensed treatments or vaccines available for these viruses in humans. Experimental vaccines have shown variable efficacy and may cause severe adverse effects. Here, we outline recent strategies used to generate vaccines against EEEV, VEEV, and WEEV with an emphasis on virus-vectored and plasmid DNA delivery. Despite candidate vaccines protecting against one of the three viruses, few studies have demonstrated an effective trivalent vaccine. We evaluated the potential of published vaccines to generate cross-reactive protective responses by comparing DNA vaccine sequences to a set of EEEV, VEEV, and WEEV genomes and determining the vaccine coverages of potential epitopes. Finally, we discuss future directions in the development of vaccines to combat EEEV, VEEV, and WEEV.

Exploring the Biocompatibility of Near-IR CuInSexS2-x/ZnS Quantum Dots for Deep-Tissue Bioimaging.

Near-infrared (NIR) emitting quantum dots (QDs) with emission in the biological transparency windows (NIR-I: 650-950 nm and NIR-II: 1000-1350 nm) are promising candidates for deep-tissue bioimaging. However, they typically contain toxic heavy metals such as cadmium, mercury, arsenic, or lead. We report on the biocompatibility of high brightness CuInSexS2-x/ZnS (CISeS/ZnS) QDs with a tunable emission covering the visible to NIR (550-1300 nm peak emission) and quantify the transmission of their photoluminescence through multiple biological components to evaluate their use as imaging agents. In general, CISeS/ZnS QDs were less cytotoxic to mouse fibroblast cells when compared with commercial CdSe/ZnS and InP/ZnS QDs. Surprisingly, InP/ZnS QDs significantly upregulated expression of apoptotic genes in mouse fibroblast cells, while cells exposed to CISeS/ZnS QDs did not. These findings provide insight into biocompatibility and cytotoxicity of CISeS/ZnS QDs that could be used for bioimaging.

Direct detection of bacteremia by exploiting host-pathogen interactions of lipoteichoic acid and lipopolysaccharide.

Bacteremia is a leading cause of death in sub-Saharan Africa where childhood mortality rates are the highest in the world. The early diagnosis of bacteremia and initiation of treatment saves lives, especially in high-disease burden areas. However, diagnosing bacteremia is challenging for clinicians, especially in children presenting with co-infections such as malaria and HIV. There is an urgent need for a rapid method for detecting bacteremia in pediatric patients with co-morbidities to inform treatment. In this manuscript, we have developed and clinically validated a novel method for the direct detection of amphiphilic pathogen biomarkers indicative of bacteremia, directly in aqueous blood, by mimicking innate immune recognition. Specifically, we have exploited the interaction of amphiphilic pathogen biomarkers such as lipopolysaccharides (LPS) from Gram-negative bacteria and lipoteichoic acids (LTA) from Gram-positive bacteria with host lipoprotein carriers in blood, in order to develop two tailored assays - lipoprotein capture and membrane insertion - for their direct detection. Our assays demonstrate a sensitivity of detection of 4 ng/mL for LPS and 2 ng/mL for LTA using a waveguide-based optical biosensor platform that was developed at LANL. In this manuscript, we also demonstrate the application of these methods for the detection of LPS in serum from pediatric patients with invasive Salmonella Typhimurium bacteremia (n = 7) and those with Staphylococcal bacteremia (n = 7) with 100% correlation with confirmatory culture. Taken together, these results demonstrate the significance of biochemistry in both our understanding of host-pathogen biology, and development of assay methodology, as well as demonstrate a potential new approach for the rapid, sensitive and accurate diagnosis of bacteremia at the point of need.

Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone.

Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans, plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presence of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.

Presentation matters: Impact of association of amphiphilic LPS with serum carrier proteins on innate immune signaling.

Recognition of Pathogen-associated Molecular Patterns (PAMPs) by Toll-like receptors is central to innate immunity. Many bacterial PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid have amphiphilic properties. The hydrophobicity of amphiphilic PAMPs contributes to increasing entropy and causes these molecules to self-aggregate or bind host carrier proteins in aqueous physiological environments. The goal of this work was to determine how innate immune signaling is impacted by physical presentation and association of amphiphilic PAMPs with serum carrier proteins, using LPS as an example molecule. Specifically, we measured LPS-induced cytokine profiles in murine macrophages when the antigen was presented associated with the various serum carrier proteins in serum versus a serum-depleted system. Our study demonstrates that the observed cytokine profiles are dramatically different when LPS is presented in buffer, versus in serum when it is associated with proteins, specifically with respect to inhibition of pro-inflammatory cytokines in the latter. These studies suggest that LPS-mediated cytokine expression is dependent on its presentation in physiological systems. The amphiphilicity of bacterial PAMPs and consequent association with lipoproteins is a feature, which should be taken into account in the design of in vitro experiments. Further studies of the interdependencies of different serum carriers can identify pathways for drug delivery and diagnostics.

Transfer and Persistence of a Multi-Drug Resistance Plasmid in situ of the Infant Gut Microbiota in the Absence of Antibiotic Treatment.

The microbial ecosystem residing in the human gut is believed to play an important role in horizontal exchange of virulence and antibiotic resistance genes that threatens human health. While the diversity of gut-microorganisms and their genetic content has been studied extensively, high-resolution insight into the plasticity, and selective forces shaping individual genomes is scarce. In a longitudinal study, we followed the dynamics of co-existing Escherichia coli lineages in an infant not receiving antibiotics. Using whole genome sequencing, we observed large genomic deletions, bacteriophage infections, as well as the loss and acquisition of plasmids in these lineages during their colonization of the human gut. In particular, we captured the exchange of multidrug resistance genes, and identified a clinically relevant conjugative plasmid mediating the transfer. This resistant transconjugant lineage was maintained for months, demonstrating that antibiotic resistance genes can disseminate and persist in the gut microbiome; even in absence of antibiotic selection. Furthermore, through in vivo competition assays, we suggest that the resistant transconjugant can persist through a fitness advantage in the mouse gut in spite of a fitness cost in vitro. Our findings highlight the dynamic nature of the human gut microbiota and provide the first genomic description of antibiotic resistance gene transfer between bacteria in the unperturbed human gut. These results exemplify that conjugative plasmids, harboring resistance determinants, can transfer and persists in the gut in the absence of antibiotic treatment.

Detection of Lipid and Amphiphilic Biomarkers for Disease Diagnostics.

Rapid diagnosis is crucial to effectively treating any disease. Biological markers, or biomarkers, have been widely used to diagnose a variety of infectious and non-infectious diseases. The detection of biomarkers in patient samples can also provide valuable information regarding progression and prognosis. Interestingly, many such biomarkers are composed of lipids, and are amphiphilic in biochemistry, which leads them to be often sequestered by host carriers. Such sequestration enhances the difficulty of developing sensitive and accurate sensors for these targets. Many of the physiologically relevant molecules involved in pathogenesis and disease are indeed amphiphilic. This chemical property is likely essential for their biological function, but also makes them challenging to detect and quantify in vitro. In order to understand pathogenesis and disease progression while developing effective diagnostics, it is important to account for the biochemistry of lipid and amphiphilic biomarkers when creating novel techniques for the quantitative measurement of these targets. Here, we review techniques and methods used to detect lipid and amphiphilic biomarkers associated with disease, as well as their feasibility for use as diagnostic targets, highlighting the significance of their biochemical properties in the design and execution of laboratory and diagnostic strategies. The biochemistry of biological molecules is clearly relevant to their physiological function, and calling out the need for consideration of this feature in their study, and use as vaccine, diagnostic and therapeutic targets is the overarching motivation for this review.

Genome Dynamics of Escherichia coli during Antibiotic Treatment: Transfer, Loss, and Persistence of Genetic Elements In situ of the Infant Gut.

Elucidating the adaptive strategies and plasticity of bacterial genomes in situ is crucial for understanding the epidemiology and evolution of pathogens threatening human health. While much is known about the evolution of Escherichia coli in controlled laboratory environments, less effort has been made to elucidate the genome dynamics of E. coli in its native settings. Here, we follow the genome dynamics of co-existing E. coli lineages in situ of the infant gut during the first year of life. One E. coli lineage causes a urinary tract infection (UTI) and experiences several alterations of its genomic content during subsequent antibiotic treatment. Interestingly, all isolates of this uropathogenic E. coli strain carried a highly stable plasmid implicated in virulence of diverse pathogenic strains from all over the world. While virulence elements are certainly beneficial during infection scenarios, their role in gut colonization and pathogen persistence is poorly understood. We performed in vivo competitive fitness experiments to assess the role of this highly disseminated virulence plasmid in gut colonization, but found no evidence for a direct benefit of plasmid carriage. Through plasmid stability assays, we demonstrate that this plasmid is maintained in a parasitic manner, by strong first-line inheritance mechanisms, acting on the single-cell level, rather than providing a direct survival advantage in the gut. Investigating the ecology of endemic accessory genetic elements, in their pathogenic hosts and native environment, is of vital importance if we want to understand the evolution and persistence of highly virulent and drug resistant bacterial isolates.