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Pathogenomics Of Non-pathogens

Staphylococcus epidermidis is generally a relatively benign resident of human skin and mucosal surfaces. However, over recent decades, the increased use of invasive devices and implants, such as catheters, has contributed to the rise of this bacterium as a hospital-acquired pathogen. The severity and diversity of the infections caused by this pathogen are not as great as those associated with its more notorious cousin Staphylococcus aureus, but its ability to form biofilms on artificial surfaces means that it is often recalcitrant to antimicrobial therapy and therefore difficult to treat.

S. Epidermidis strain ATCC 12228 is a non-biofilm-forming, non-infection-associated isolate that has previously been used for antibiotic detection in food products. In their publication, Zhang et al.1 present the complete genome sequence, and use previously published genome sequences of clinical S. Aureus isolates2,3 to identify components of the ATCC 12228 genome involved in virulence, drug resistance and regulation. The authors found that the ATCC 12228 genome contains fewer virulence determinants than the clinical S. Aureus isolates — it lacks orthologues of pyrogenic toxins with superantigen activity, and the only toxins identified were the membrane-active β- and δ-haemolysins. This strain also lacks the virulence-associated extracellular enzymes staphylokinase, staphylocoagulase and hyaluronidase. It is better equipped with genes encoding adhesins: in addition to previously characterized S. Epidermidis adhesin genes (altE, fbe and embP), the genome contains orthologues of several S. Aureus MSCRAMMs that have been shown to modulate host-cell interactions. Although the ATCC 12228 isolate therefore appears to be well endowed with potential adhesins capable of binding specific host molecules, it does not contain the ica operon, which is required for polysaccharide-mediated adhesion and biofilm formation in some strains of S. Epidermidis. The region of the ATCC 12228 chromosome that is similar to the flanking regions of the ica operon appears to contain signs of genetic rearrangements, leading the authors to suggest that the absence of this operon is the result of a genetic defect.

The comparison of this genome with those of the clinical S. Aureus isolates reveals that this strain's virulence arsenal is not as well stocked. However, as this strain is a non-clinical isolate with low virulence in an animal model, the real significance of the arsenal's remaining contents in virulence and colonization is unclear. The apparent loss of the ica operon also raises the question as to whether any virulence functions, or indeed additional biofilm genes, could also be missing. To this end, the publication of the complete genome of the biofilm-forming, clinically isolated S. Epidermidis strain RP62A is keenly awaited.

The genome sequence of Chromobacterium violaceum ATCC 12472 reveals a 4.75 Mb single chromosome with 65% G+C content4. This unusual free-living microorganism was isolated from a tropical soil and water environment. Approximately one-third of the predicted genes encode transport proteins, which are thought to be highly specific and permit efficient scavenging of very low concentrations of nutrients. C. Violaceum can adapt quickly to harsh environmental stress using alternative metabolic pathways for growth depending on the nutrients available. Tight transcriptional control is achieved by many regulators, including several sigma factors. The organism produces violacein — a purple pigment with antimicrobial properties against major pathogens such as Mycobacterium tuberculosis — and its production is under quorum-sensing control. The considerable biotechnological potential of C. Violaceum has been explored with its use in methods to synthesize polyhydroxyalkanoates (PHAs), hydrolyze plastic films and solubilize gold without mercury. C. Violaceum occasionally infects humans but the genome has no obvious pathogenicity islands; it is suggested that these could be isolate-specific. However, there is an almost-complete type III secretion system and some genes similar to those found in Salmonella typhimurium invasion loci. Insecticidal, nematocidal and antibiotic synthesis genes are also present, giving some indication of the diversity of the environment inhabited by this organism.

Finally, Gloeobacter violaceus PCC 7421 is a unicellular cyanobacterium whose genome consists of a single chromosome of just over 4.5 Mb5. Key differences have been found in its genome that set this bacterium apart from the rest of the cyanobacterial group it belongs to; this reflects a considerable phylogenetic distance as the Gloeobacter lineage was the earliest to diverge within the radiation of cyanobacteria and chloroplasts. One of the main differences reflects an ancestral characteristic: G. Violaceus has the necessary genes for photosystems I and II, which are both required for oxygenic photosynthesis6, however this process occurs in the cytoplasmic membrane and not in the thylakoid membrane, as thylakoids are not present in G. Violaceus. Therefore, although the photosynthetic structures present in other cyanobacteria face the lumen of the thylakoids, in this bacterium they face the periplasmic space and hence co-exist with the respiratory system. The other main difference is related to circadian rhythm. G. Violaceus lacks the major genetic elements of the circadian clock, the kai genes, which are involved in controlling cell division in other cyanobacteria, suggesting that this system could have been acquired after the divergence of the Gloeobacter lineage.

Fighting Invisible Foes: How Cryptic Fungal Pathogens Threaten Human Health And Food Security

In a recent study published in the journal PLOS Pathogens, researchers discussed cryptic fungal pathogens, which are genetically distinct from pathogens but morphologically indistinguishable.

Fungal pathogens threaten global health and food security. Diagnosing and treating fungal infections are complicated, leading to severe illness and death. Besides, cryptic fungal species can also cause infections, and their identification through conventional methods is challenging due to morphological similarities. As such, cryptic fungi's clinical burden and epidemiology are poorly understood. The present study summarized the available information on cryptic fungi.

Study: Know the enemy and know yourself: Addressing cryptic fungal pathogens of humans and beyond. Image Credit: Created with the assistance of DALL·E 3

Diagnosis of fungal pathogens

Accurate diagnosis is essential to decrease the burden of fungal infections. Conventional diagnosis methods include microscopy, histopathology, culture, and matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry. These methods have poor specificity, particularly for cryptic species. Nevertheless, molecular typing (phylogenetic analysis) has been more accurate.

However, some loci may not have sufficient information for accurate detection. Aspergillus latus is a cryptic species and allodiploid hybrid that has remained undetected in clinical settings due to the limitations of single-locus typing in a hybrid genome. Extensive phenotyping and genome sequencing have provided robust detection of A. Latus. Other approaches, such as phylogenomics, can overcome these limitations.

Although phylogenomics offers high specificity, it requires more time, resources, and expertise. Rapid and affordable diagnosis is critical to lower mortality. Of note, average nucleotide identity is an unexplored alternative. A comparative analysis of various diagnostic methods would determine the most effective method.

Exemplary known and cryptic pathogenic species of Aspergillus. (Left) Aspergillus fumigatus is a well-known and major human fungal pathogen. The cryptic species (Middle) Aspergillus lentulus and (Right) Aspergillus udagawae can also cause human disease but may be difficult to diagnose owing to their morphological similarity to A. fumigatus. Images were kindly provided by Dr. Jos Houbraken. https://doi.Org/10.1371/journal.Ppat.1011704.G001

Human influence

Humans may unwittingly contribute to the transmission of fungal diseases. For instance, invasive fungal outbreaks have been linked to hospital construction, poor air filtration, and spread through contaminated surfaces. Besides, dietary effects on the abundance of specific fungi in the human mycobiome, human-to-human transmission, and antifungal resistance can also impact the fungal disease.

Examining patient populations and their genetic backgrounds will be critical to understanding how host biology contributes to susceptibility. That is, chronic granulomatous disease patients have a higher risk of infection with A. Nidulans. Notably, the associations between cryptic fungal pathogens and specific patient populations are unclear due to the lack of precise diagnosis.

Immune response and disease management

Understanding the interactions of fungal pathogens with the host immune system will provide critical insights into their persistence and clearance mechanisms. Some studies have identified immunological checkpoints for pathogen recognition. Moreover, there is substantial heterogeneity in the formation of neutrophil extracellular traps, i.E., NETosis, across Aspergillus species and strains.

Furthermore, some congenital abnormalities in immune functions may increase the susceptibility to infection in specific populations. For example, individuals with chronic granulomatous disease exhibit higher susceptibility to aspergillosis as they have a lower capacity to produce reactive oxygen species (ROS).

Influence of microbiome and potential future threats

Exploring the human microbiome can help delineate species' impact in cross-species interactions that may prevent or contribute to disease. Mycobiome alterations may contribute to disease severity. Pathogens also impact the mycobiome; that is, lung colonization by A. Fumigatus results in microbiome composition being more favorable for fungal growth.

Although studies on the mycobiome have offered vital insights at the genus level, assessing variations at the species or strain level will deepen the understanding of how they influence human health and mycobiome. Cryptic pathogens are often undetected, as identification is possible only at the genus level.

Therefore, species or strain level identification is fundamental, given the phenotypic and genotypic heterogeneity. Analysis of non-pathogenic species is also essential, as previously unrecognized species could emerge as pathogens. Exploring pathogens and related non-pathogenic species can provide insights into the emergence of pathogenicity and evolutionary dynamics.

Concluding remarks

Addressing these challenges requires establishing essential infrastructure for accurate diagnosis and dissemination of results. This will facilitate understanding the epidemiology, disease surveillance, and identifying susceptible populations, early outbreaks, and emerging pathogens. Insights from surveillance systems, such as Nextstrain and Microreact, can serve as an inspiration to establish a fungal-specific platform. Overall, integrating microbiome, pathogen, host genetics, and health records data will provide a comprehensive dataset to address the concerns of fungal pathogens.

Non-tuberculous Mycobacteria

The author reported a case of mycobacterial infection, which was diagnosed after a lengthy process.

However, the case is actually not one of tuberculosis in the stricter sense: the confirmed species—Mycobacterium avium intracellulare complex—belongs to the group of non-tuberculous mycobacteria. These are often found in the environment and, in cultures, are possibly only contaminants. However, in this case the diagnosis was firmly established following the criteria of the American Thoracic Society (ATS) – the specimens came from otherwise sterile body fluids (1).

Microscopy using different staining methods, as mentioned in the article, is less relevant than cultures and PCR for confirming a diagnosis, since microscopy has lower sensitivity and, in contrast to the other methods, cannot differentiate between species (2). It therefore needs to be emphasized that the diagnostic mistake in the case report consisted mainly in not doing culture and PCR.

Non-tuberculous mycobacteria—such as the described species—display sensitivities to anti-mycobacterial chemotherapy different from Mycobacterium tuberculosis complex. After resistance testing, recommended initial treatment should therefore consist of a combination of clarithromycin, ethambutol, and, if required, rifabutin. By contrast to the treatment described in the article, however, isoniazid is not the treatment of choice (3).DOI: 10.3238/arztebl.2010.0147b

Dr. Med. Norbert HeinrichPD Dr. Med. Michael HoelscherAbteilung für Infektions- und TropenmedizinKlinikum der LMULeopoldstr. 580802 München, Germanyheinrich@lrz.Uni-muenchen.De

PD Dr. Rer. Nat. Elvira RichterForschungszentrum BorstelNationales Referenzzentrum fürMykobakterienParkallee 1823845 Borstel, Germany

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