Staphylococcus versus Staphylococcus

Antibiotics right under our noses

Andrey Panov, "Biomolecule"

German scientists have discovered a new weapon to fight the hospital monster – multi-resistant Staphylococcus aureus. For many years it has been hiding not in the permafrost or the Mariana Trench, but right under our noses. Or rather, in it.

In recent years, the number of infectious diseases caused by antibiotic-resistant bacteria has increased worldwide. Organisms with multidrug-resistant organisms (MDRO), such as methicillin-resistant Staphylococcus aureus, enterococci insensitive to vancomycin or gram-negative bacteria resistant to cephalosporins of the third generation, may become more frequent causes of death than cancer in the coming decades [1].

Antibiotics available to the population are losing their effectiveness, and their frequent and unjustified use leads to the selection of resistant forms of bacteria inside human and animal organisms. MDRO in the human microbiome is difficult to detect, because usually their carriage is asymptomatic. However, under stressful conditions (after surgery, injury or immunosuppression) it can develop into an aggressive infection, which will be extremely difficult to overcome. And if we also take into account the frequent resistance of such bacteria to classical disinfectants, it becomes clear why MDRO is considered a real scourge of hospitals and maternity hospitals. They are a much more tangible threat to the population of all continents than the same Ebola virus, so huge efforts are now being directed to the search and production of new antibiotics that can "crack" the MDRO defense systems.

A few years ago, it was discovered that representatives of the human microbiota are able to produce bacteriocins that affect closely related bacteria [2]. For example, in 2014, a new thiopeptide antibiotic, lactocillin, synthesized by ribosomes, was isolated and described from the human commensal Lactobacillus gasseri [3].

Biomolecula spoke in detail about peptide antibiotics earlier: "Antimicrobial peptides are a possible alternative to traditional antibiotics" [4].

It may seem strange that the human microbiota produces antibiotics, because the antimicrobial search industry has always been focused on soil bacteria: it was believed that it was there that life was raging and the struggle for existence was actively going on. However, in the human microbiome there are more than a thousand species of bacteria competing for space and nutrients. This contributes to the appearance of a real "weapon of mass destruction" – bacteriocins produced with the help of two types of enzymes – polyketide synthases and non-ribosomal peptidesynthetases [5].

Family showdowns

In July 2016, German researcher Alexander Zipperer and his collaborators reported that they had discovered the bacterium Staphylococcus lugdunensis IVK28 in the human nose, which suppresses the growth of methicillin-resistant Staphylococcus aureus (MRSA) [1].

Staphylococcus aureus

Staphylococci are typical commensal bacteria that colonize the skin and the surfaces of mucous membranes. Staphylococcus aureus are globular gram–positive bacteria that produce a carotenoid pigment that gives their cells a golden color (Fig. 1). These microorganisms are extremely resistant to external influences and survive in the air, dust, soil, food, food production equipment and household items [6, 7].

Figure 1. Staphylococcus aureus and leukocytes. Drawing from the website relatedscience.blogspot.ru .

Staphylococcus aureus is a conditionally pathogenic bacterium that exhibits its pathological properties only under favorable conditions, and creates them, as a rule, by weakening the immunity of the carrier. Active vital activity of staphylococcus can lead to various diseases [5, 7, 8]:

• skin (pimples, boils, scalded skin syndrome);
• respiratory organs (pleurisy, pneumonia);
• bone and connective tissues (arthritis, osteomyelitis);
• nervous system and sensory organs (otitis media, meningitis);
• cardiovascular system (endocarditis, phlebitis, staphylococcal bacteremia).

The pathogenicity factors of S.aureus are microcapsule, cell wall components, aggression enzymes and toxins. Microcapsules protect bacterial cells from phagocytosis, promote their adhesion and spread throughout the host body. The components of the cell wall (for example, peptidoglycan, teichoic acids and protein A) cause the development of inflammation, immobilize phagocytes and neutralize immunoglobulins. Coagulase, the main enzyme of aggression, causes blood plasma coagulation [7, 9].

Methicillin-resistant staphylococci (methicillin-resistant S.aureus, MRSA) are the most dangerous (Fig. 2). Methicillin is a modified penicillin, with which they have recently successfully fought against staphylococcal infection. MRSA is resistant not only to methicillin, but also to other antibiotics of the penicillin group (dicloxacillin, oxacillin, nafcillin, etc.), as well as to cephalosporins.

Figure 2 (from the website thinglink.com ). Methicillin-resistant Staphylococcus aureus.

Recently, strains with a wider spectrum of resistance have also been identified: vancomycin-resistant (VRSA) and glycopeptide-resistant (GISA) [6, 9, 10].

The main sources of infection with Staphylococcus aureus are patients with an erased form of infection and asymptomatic carriers. The greatest danger comes from medical personnel: according to some data, the carrier among physicians can reach 35% and, in comparison with the main population, they are much more often "populated" with antibiotic-resistant strains. S.aureus can be transmitted through the hands of medical staff and non-sterile medical instruments, when using intravenous catheters and artificial lung ventilation. After hospitalization, 20-30% of patients taking antibiotics, diabetic patients or undergoing hemodialysis become carriers of Staphylococcus aureus [7, 10]. This is also why competent doctors call for a deliberate approach to hospitalization – to resort to it only in case of real need and to be discharged as soon as possible. In addition to Staphylococcus and other potentially multi–resistant bacteria, very unpleasant viruses - rota- and noro- are rapidly spreading among patients, visitors and staff there, but it is rarely considered necessary to warn about hospital outbreaks of "toilet" infections. Therefore, "go lie down for prevention, dig through" may turn out to be a little unexpected side...

The S.lugdunensis IVK28 strain effectively fought its harmful relative only under conditions of iron deficiency and only on solid agarized media (Fig. 3, left). The mechanism of the confrontation was unclear, and therefore Zipperer performed transposon mutagenesis of cells of the isolated strain – to identify the gene responsible for the synthesis of a deadly substance for S.aureus.

Figure 3. Antibacterial activity of S.lugdunensis against methicillin-resistant S.aureus. On the left – wild strain IVK28 forms a lysis zone on S.aureus culture. In the center, the strain IVK28ΔlugD (with the transposon insertion of the lugD gene "turned off") has no effect on Staphylococcus aureus. On the right – a strain with restored lugD gene activity again lyzes competitor cells. Figure from [1].

As a result, it was possible to obtain a mutant IVK28, which could not suppress the growth of MRSA. Analysis of the transposon insertion site showed that it disrupted the structure of the gene of the putative non-ribosomal peptide synthetase (NRPS). It turned out that this gene, along with other sequences associated with the biosynthesis of antibiotics, is part of an operon with a size of 30 t.n. This indicated that the proposed inhibitor molecule may be a complex of non-ribosomal peptides.

The operon was detected by PCR in all cultures of S.lugdunensis, which means that it is characteristic of the entire species, and not only for the IVK28 strain. However, the GC composition of the operon (26.9%) differed from the GC composition of the rest of the S.lugdunensis genome (33.8%), which indicated the possible borrowing of this useful genetic cluster from other bacterial species by horizontal transfer.

The diverse participants and piquant details of bacterial horizontal genetic transfer are described in the article "Mobile genetic elements of prokaryotes: stratification of the "society of "vagrants and stay-at-home people" [11].

The operon consists of the lugA, B, C and D genes encoding peptide synthetase proteins (see box below), as well as other genes whose products are necessary for the synthesis and transport of a non-ribosomal peptide.

In order to finally impute the operon participation in the antibacterial activity of S.lugdunensis, the smallest gene (lugD) was removed. The ΔlugD mutant, as expected, could not suppress the growth of Staphylococcus aureus, but when a plasmid with a working lugD gene was injected into it, the aggressive phenotype was restored (Fig. 3, center and right).

Secret Weapon

The lug operon product isolated by Zipperer turned out to be a non-ribosomal cyclic peptide consisting of five amino acids (two D-valines, L-valine, D-leucine and L-tryptophan) and a thiazolidine heterocycle (Fig. 4). The antibiotic was called lugdunin.

Figure 4. Gene cluster, biosynthetic pathway and chemical structure of lugdunin. a – Genes of "subunits" (not modules!) non–ribosomal peptide synthetase S.lugdunensis: lugA, B, C and D. b - Functional domains of operon products: A – adenylating, P – peptidyl, C – condensing, E – epimerizing, R – reductase. Their specific combinations make up modules – isolated catalytic units of the enzyme. Lugdunin biosynthesis begins, apparently, in the initiating LugD module and continues sequentially with LugA-C. b is the structural formula of lugdunin. Figure from [1].

By chemical synthesis, it was possible to obtain a product with chemical properties identical to natural lugdunin and antibacterial effect. Scientists have suggested that this antibiotic inhibits the synthesis of bacterial biopolymers – proteins, DNA and peptidoglycans [5].

Non - ribosomal peptides

This class of peptides is synthesized in the cells of lower fungi and bacteria without the participation of ribosomes. Non-ribosomal peptides (NRPS) are also found in higher organisms that have commensal bacteria [12].

NRPS are divided into several functional groups [13]:

• antibiotics (vancomycin);
• precursors of antibiotics (ACV-tripeptide – precursor of penicillin and cephalosporin);
• immunosuppressants (cyclosporine);
• antitumor peptides (bleomycin);
• siderophores (pioverdin);
• toxins (HC-toxin);
• surfactants (surfactin).

Building

Non-ribosomal peptides range in length from 2 to 50 amino acids and often have a cyclic or branched structure. They contain both "ordinary", proteinogenic and non–proteinogenic amino acids - D-forms or residues modified by addition of N-methyl and N-formyl groups, glycosylation, hydroxylation, acylation or halogenation. Cyclization occurs by the formation of oxazolines and thiazolines in the peptide backbone [12].

Synthesis

NRPS are synthesized by non-ribosomal peptide synthetases (NRPS), which do not follow "foreign" instructions in their work, that is, they do without mRNA. HPPs are giant multimodule enzymes, each of which can synthesize only one type of peptides. A separate module of the enzyme is responsible for the inclusion of one amino acid in the peptide chain, so the number of modules corresponds to the length of the peptide [14].

Each module consists of at least three domains:

• condensing (receiving the peptide chain from the previous module);
• adenylating (choosing the right amino acid);
• peptidyl (forming a peptide bond).

Modules often include other domains, including an epimerizing domain that converts L-amino acids into D-forms [14].

By analogy with the triplet ribosomal code for protein synthesis, there is also a non-ribosomal, NPS code defined by 10 amino acid residues in the substrate-binding pocket of the adenylating domain. The combination of these residues determines which amino acid will be embedded in the peptide by a specific NPS module. Knowing this code, it is possible to predict the substrate specificity of adenylating domains and even arbitrarily change it by replacing amino acids in the domain [14].

In the experiments of German scientists, lugdunin acted not only on methicillin-resistant staphylococci, but also on glycoprotein-resistant, and even on other gram-positive bacteria such as listeria and vancomycin–resistant enterococcus (Table 1). The minimum inhibitory concentration (MIC) of the new bacteriocin is 1.5–12 mcg × ml⁻¹, which says about the high activity of the substance. At the same time, such concentrations did not affect human serum in any way, did not cause neutrophil or erythrocyte lysis, and did not inhibit the metabolic activity of monocytes. Bacterial cells, however, stopped synthesizing DNA, RNA, proteins and cell wall components under the action of lugdunin in concentrations even below MIC. In this respect, lugdunin resembles daptomycin, which gives the same effect, but the mode of action of which has not yet been studied. There was no evidence of resistance of S.aureus cells to lugdunin even after their monthly cultivation at low concentrations.

Tests in combat conditions

As expected, lugdunin's ability to treat staphylococcal infections was demonstrated in vivo on a mouse model (Fig. 5). In six mice, the hair on the back was shaved off and, after damaging the skin by repeatedly gluing / peeling off the patch, Staphylococcus aureus was applied to this place. Then the skin was treated with an ointment containing 1.5 micrograms of lugdunin, and six hours later the result was evaluated. Treatment with a new antibiotic greatly reduced or even completely destroyed the S.aureus population. And not only on the surface of the skin, but also in its deeper layers.

Figure 5. General scheme of the approach to the identification of a natural antibiotic. Representatives who cannot coexist with pathogenic bacteria of interest are selected from bacterial populations of the human body. These possible competitors are tested separately on media with an infectious agent. An antibiotic is isolated from a culture that successfully suppresses the growth of pathogens, the effect of which is tested on animal models. Figure from [5], modified and adapted.

To understand whether S.lugdunensis can prevent colonization of the nasal cavity of vertebrates with Staphylococcus aureus in vivo, scientists conducted the following experiment. Two types of mixed cultures (S.aureus + S.lugdunensis IVK28 and S.aureus + S.lugdunensis IVK28ΔlugD) were introduced into the noses of cotton hamsters and each separately. In control cases, when one strain was injected, all three cultures stably colonized the nasal cavity. However, with the introduction of a mixture of S.aureus + S.lugdunensis IVK28, the amount of Staphylococcus aureus after 5 days significantly decreased compared to the mixture of S.aureus + S.lugdunensis IVK28ΔlugD. This experiment showed that lugdunin production allows the IVK28 strain to compete effectively with Staphylococcus aureus in vivo.

It remained to figure out whether the presence of S.lugdunensis in the human nose prevents colonization by S.aureus bacteria. Zipperer and his colleagues examined smears from the nasal passages of 187 hospitalized patients. Of these, 60 people (32.1%) were found to have Staphylococcus aureus and 17 people (9.1%) had S.lugdunensis. And only one patient with S.lugdunensis had S.aureus in his nose. In all isolated S.lugdunensis strains, PCR analysis demonstrated the presence of a lug operon, and all detected S.aureus strains were susceptible to lugdunin.

The prospects

Due to the high effectiveness of lugdunin, the authors of the work under discussion suggest using S.lugdunensis in the fight against Staphylococcus aureus, especially in patients with high risks of infection – after surgery, immunosuppression or hemodialysis. Previously, probiotic, as a rule, were called bacteria that actively act for the benefit of the macroorganism in the gastrointestinal tract. The Zipperer group advocates expanding the concept of "probiotics" to include bacteria that fight infections in other parts of the human body, such as the nasal cavity or skin.

In very rare cases, S.lugdunensis itself can cause diseases, but if it is possible to create mutants that have completely lost virulence factors, or to embed a lug operon in absolutely "peaceful" bacteria, a safe probiotic drug can be developed.

Lugdunin was the first discovered bacteriocin of a new class – macrocyclic thiazolidine peptide antibiotics. All tested strains of S.aureus (both natural and laboratory) failed to develop resistance to it. This gives hope that lugdunin will become a commercial drug for the fight against Staphylococcus aureus in the future.

And finally, the very fact of the discovery of a new antibiotic in a representative of the human microbiota should serve as an incentive to intensify the search for other producers of bacteriocins in the composition of such communities. In the future, this will help doctors to successfully restrain the onset of multi-resistant pathogens.

Literature

1. Zipperer A., Konnerth M.C., Laux C., Berscheid A., Janek D., Weidenmaier C. et al. (2016). Human commensals producing a novel antibiotic impair pathogen colonization. Nature. 535, 511–516;
2. Dobson A., Cotter P.D., Ross R.P., Hill C. (2012). Bacteriocin production: a probiotic trait? Appl. Environ. Microbiol. 78, 1–6;
3. Donia M.S., Cimermancic P., Schulze C.J., Wieland Brown L.C., Martin J., Mitreva M. et al. (2014). A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. Cell. 158, 1402–1414;
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14. Mironovskiĭ M.L., Ostash B.E., Fedorenko V.A. (2010). Diversity of genes encoding nonribosomal peptide synthetases in the Streptomyces sioyaensis genome. Genetika. 46, 896–903.

Portal "Eternal youth" http://vechnayamolodost.ru  25.08.2016