Detection of Vancomycin Resistant Genes in Intrinsically Antibiotic Resistant Bacteria from the Gut Microbiota of Indonesian Individuals.

Background: Antibiotic resistance is a global public health concern that has been exacerbated by the overuse and misuse of antibiotics, leading to the emergence of resistant bacteria. The gut microbiota, often influenced by antibiotic usage, plays a crucial role in overall health. Therefore, this study aimed to investigate the prevalence of antibiotic resistant genes in the gut microbiota of Indonesian coastal and highland populations, as well as to identify vancomycin-resistant bacteria and their resistant genes. Methods: Stool samples were collected from 22 individuals residing in Pacet, Mojokerto, and Kenjeran, Surabaya Indonesia in 2022. The read count of antibiotic resistant genes was analyzed in the collected samples, and the bacterium concentration was counted by plating on the antibiotic-containing agar plate. Vancomycin-resistant strains were further isolated, and the presence of vancomycin-resistant genes was detected using a multiplex polymerase chain reaction (PCR). Results: The antibiotic resistant genes for tetracycline, aminoglycosides, macrolides, beta-lactams, and vancomycin were found in high frequency in all stool samples (100%) of the gut microbiota. Meanwhile, those meant for chloramphenicol and sulfonamides were found in 86% and 16% of the samples, respectively. Notably, vancomycin-resistant genes were found in 16 intrinsically resistant Gram-negative bacterial strains. Among the detected vancomycin-resistant genes, vanG was the most prevalent (27.3%), while vanA was the least prevalent (4.5%). Conclusion: The presence of multiple vancomycin resistance genes in intrinsically resistant Gram-negative bacterial strains demonstrated the importance of the gut microbiota as a reservoir and hub for the horizontal transfer of antibiotic resistant genes.

From an Hsp90 - binding protein to a peptide drug.

The Lpl proteins represent a class of lipoproteins that was first described in the opportunistic bacterial pathogen Staphylococcus aureus, where they contribute to pathogenicity by enhancing F-actin levels of host epithelial cells and thereby increasing S. aureus internalization. The model Lpl protein, Lpl1 was shown to interact with the human heat shock proteins Hsp90α and Hsp90ß, suggesting that this interaction may trigger all observed activities. Here we synthesized Lpl1-derived peptides of different lengths and identified two overlapping peptides, namely, L13 and L15, which interacted with Hsp90α. Unlike Lpl1, the two peptides not only decreased F-actin levels and S. aureus internalization in epithelial cells but they also decreased phagocytosis by human CD14+ monocytes. The well-known Hsp90 inhibitor, geldanamycin, showed a similar effect. The peptides not only interacted directly with Hsp90α, but also with the mother protein Lpl1. While L15 and L13 significantly decreased lethality of S. aureus bacteremia in an insect model, geldanamycin did not. In a mouse bacteremia model L15 was found to significantly decreased weight loss and lethality. Although the molecular bases of the L15 effect is still elusive, in vitro data indicate that simultaneous treatment of host immune cells with L15 or L13 and S. aureus significantly increase IL-6 production. L15 and L13 represent not antibiotics but they cause a significant reduction in virulence of multidrug-resistant S. aureus strains in in vivo models. In this capacity, they can be an important drug alone or additive with other agents.

The orchestra of human bacteriome by hormones.

Human microbiome interact reciprocally with the host. Recent findings showed the capability of microorganisms to response towards host signaling molecules, such as hormones. Studies confirmed the complex response of bacteria in response to hormones exposure. These hormones impact many aspects on bacteria, such as the growth, metabolism, and virulence. The effects of each hormone seem to be species-specific. The most studied hormones are cathecolamines also known as stress hormones that consists of epinephrine, norepinephrine and dopamine. These hormones affect the growth of bacteria either inhibit or enhance by acting like a siderophore. Epinephrine and norepinephrine have also been reported to activate QseBC, a quorum sensing in Gram-negative bacteria and eventually enhances the virulence of pathogens. Other hormones were also reported to play a role in shaping human microbiome composition and affect their behavior. Considering the complex response of bacteria on hormones, it highlights the necessity to take the impact of hormones on bacteria into account in studying human health in relation to human microbiome.

Identification of a serotonin N-acetyltransferase from Staphylococcus pseudintermedius ED99.

Serotonin N-acetyltransferase (SNAT) catalyzes the biosynthesis of N-acetylserotonin (NAS) and N-acetyltryptamine (NAT), two pleiotropic molecules with neurotransmitter functions. Here, we report the identification of a SNAT protein in the genus Staphylococcus. The SNAT gene identified in Staphylococcus pseudintermedius ED99, namely SPSE_0802, encodes a 140 residues-long cytoplasmic protein. The recombinant protein SPSE_0802 was expressed in E. coli BL21 and found to acetylate serotonin (SER) and tryptamine (TRY) as well as other trace amines in vitro. The production of the neuromodulators NAS and NAT was detected in the cultures of different members of the genus Staphylococcus and the role of SPSE_0802 in this production was confirmed in an ED99 SPSE_0802 deletion mutant. A search for SNAT homologues showed that the enzyme is widely distributed across the genus which correlated with the SNAT activity detected in 22 out of the 40 Staphylococcus strains tested. The N-acetylated products of SNAT are precursors for melatonin synthesis and are known to act as neurotransmitters and activate melatonin receptors, among others, inducing various responses in the human body. The identification of SNAT in staphylococci could contribute to a better understanding of the interaction between those human colonizers and the host peripheral nervous system.

Microbiome profile of soil and rhizosphere plants growing in traditional oil mining land in Wonocolo, Bojonegoro, Indonesia.

Traditional oil mining poses negative effects on the environment through pollution with crude oil. One of the traditional mining sites in Wonocolo, Bojonegoro, Indonesia was reported to contaminate the surrounding area with a high level of crude oil. Therefore, this study aims to examine the microbiome profiles of contaminated soil and the rhizosphere of naturalized plants growing at the sites. It was conducted in Wonocolo, Bojonegoro to obtain an insight into the possible remediation efforts of using indigenous hydrocarbon-degrading bacteria and naturalized plants as in situ remediation agents. The results showed that the soil located close to the oil well-contained a high level of crude oil at 24.8%, and exhibited a distinct microbiome profile compared to those located further which had lower crude oil contamination of 14.15, 10.89, and 4.9%. Soil with the highest level of crude oil contamination had a comparatively higher relative abundance of assA, an anaerobic alkene-degrading gene. Meanwhile, the rhizosphere of the two naturalized plants, Muntingia calabura, and Pennisetum purpureum, exhibited indifferent microbiome profiles compared to the soil. They were found to contain less abundant hydrocarbon-degrading genes, such as C230, PAH-RHD-GP, nahAc, assA, and alkB suggesting that these naturalized plants might not be a suitable tool for in-situ remediation.

This study provides information on the microbiome profile of soil and rhizosphere crude oil contaminated sites. The rhizosphere of growing plants in the crude-oil contaminated site exhibited a similar microbiome profile as in soil, with a lower relative abundance of hydrocarbon-degrading genes. Commonly, most inhabitant plants of the contaminated site have great potential as a phytoremediator agent, however, two largely abundant species were found to possess low potential.

Soil Mineral Composition and Salinity Are the Main Factors Regulating the Bacterial Community Associated with the Roots of Coastal Sand Dune Halophytes.

Soil salinity and mineral deficiency are major problems in agriculture. Many studies have reported that plant-associated microbiota, particularly rhizosphere and root microbiota, play a crucial role in tolerance against salinity and mineral deficiency. Nevertheless, there are still many unknown parts of plant-microbe interaction, especially regarding their role in halophyte adaptation to coastal ecosystems. Here, we report the bacterial community associated with the roots of coastal sand dune halophytes Spinifex littoreus and Calotropis gigantea, and the soil properties that affect their composition. Strong correlations were observed between root bacterial diversity and soil mineral composition, especially with soil Calcium (Ca), Titanium (Ti), Cuprum (Cu), and Zinc (Zn) content. Soil Ti and Zn content showed a positive correlation with bacterial diversity, while soil Ca and Cu had a negative effect on bacterial diversity. A strong correlation was also found between the abundance of several bacterial species with soil salinity and mineral content, suggesting that some bacteria are responsive to changes in soil salinity and mineral content. Some of the identified bacteria, such as Bacillus idriensis and Kibdelosporangium aridum, are known to have growth-promoting effects on plants. Together, the findings of this work provided valuable information regarding bacterial communities associated with the roots of sand dune halophytes and their interactions with soil properties. Furthermore, we also identified several bacterial species that might be involved in tolerance against stresses. Further work will be focused on isolation and transplantation of these potential microbes, to validate their role in plant tolerance against stresses, not only in their native hosts but also in crops.

The Ambivalent Role of Skin Microbiota and Adrenaline in Wound Healing and the Interplay between Them.

After skin injury, wound healing sets into motion a dynamic process to repair and replace devitalized tissues. The healing process can be divided into four overlapping phases: hemostasis, inflammation, proliferation, and maturation. Skin microbiota has been reported to participate in orchestrating the wound healing both in negative and positive ways. Many studies reported that skin microbiota can impose negative and positive effects on the wound. Recent findings have shown that many bacterial species on human skin are able to convert aromatic amino acids into so-called trace amines (TAs) and convert corresponding precursors into dopamine and serotonin, which are all released into the environment. As a stress reaction, wounded epithelial cells release the hormone adrenaline (epinephrine), which activates the ß2-adrenergic receptor (ß2-AR), impairing the migration ability of keratinocytes and thus re-epithelization. This is where TAs come into play, as they act as antagonists of ß2-AR and thus attenuate the effects of adrenaline. The result is that not only TAs but also TA-producing skin bacteria accelerate wound healing. Adrenergic receptors (ARs) play a key role in many physiological and disease-related processes and are expressed in numerous cell types. In this review, we describe the role of ARs in relation to wound healing in keratinocytes, immune cells, fibroblasts, and blood vessels and the possible role of the skin microbiota in wound healing.

Molecular Basis of Rhodomyrtone Resistance in Staphylococcus aureus.

Rhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory farR gene (farR*) confers resistance to Rom in Staphylococcus aureus (RomR). The mutation in farR* alters the activity of the regulator, FarR*, in such a way that not only its own gene, farR*, but also the divergently transcribed farE gene and genes controlled by the global regulator, agr, are highly upregulated. Here, we show that mainly the upregulation of the fatty acid efflux pump FarE causes the RomR phenotype, as farE deletion in either the parent or the RomR strain (RomR ΔfarE) yielded hypersensitivity to Rom. Comparative lipidome analysis of the supernatant (exolipidomics) and the pellet fraction revealed that the RomR strain excreted about 10 times more phospholipids (PGs) than the parent strain or the ΔfarE mutants. Since the PG content in the supernatant (2,244 ng/optical density [OD]) was more than 100-fold higher than that of fatty acids (FA), we assumed that PG interacts with Rom, thereby abrogating its antimicrobial activity. Indeed, by static and dynamic light scattering (SLS and DLS) and isothermal titration calorimetry (ITC) analyses, we could demonstrate that both PG and Rom were vesicular and reacted with each other in milliseconds to form a 1:1.49 [Rom-PG(32:0), where PG(32:0) is PG with C32:0 lipids] complex. The binding is entropically driven and hence hydrophobic and of low specificity in nature. Our results indicate that the cytoplasmic membrane is the actual target of Rom, which is also in agreement with Rom's induced rapid collapse of the membrane potential and decreased membrane integrity. IMPORTANCE Antibiotic resistance is a growing public health problem, and alternative antibiotics are urgently needed. Rhodomyrtone (Rom), an antimicrobial compound originally isolated from Rhodomyrtus tomentosa, is active against multidrug-resistant Gram-positive pathogens. However, Rom-resistant (RomR) mutants occur with low frequency. In this study, we unraveled the underlying resistance mechanism, which is based on a point mutation in the farR regulator gene, causing overexpression of FarE, which most likely acts as a phospholipid (PG) efflux pump, as large amounts of PG were found in the supernatant and the pellet fraction. We show that PG can bind to Rom, thereby abrogating its antimicrobial activity. The direct interaction of Rom with PG suggests that Rom's actual target is the cytoplasmic membrane. Antibiotics that interact with PG are rare. Since Rom can be chemically synthesized, it serves as a lead compound for synthesis of improved variants.

The Neuromodulator-Encoding sadA Gene Is Widely Distributed in the Human Skin Microbiome.

Trace amines (TA) are endogenously produced in mammals, have a low concentration in the central nervous system (CNS), but trigger a variety of neurological effects and intervene in host cell communication. It emerged that neurotransmitters and TA are produced also by the microbiota. As it has been shown that TA contribute to wound healing, we examined the skin microbiome of probands using shotgun metagenomics. The phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes were predominant. Since SadA is a highly promiscuous TA-producing decarboxylase in Firmicutes, the skin microbiome was specifically examined for the presence of sadA-homologous genes. By mapping the reads of certain genes, we found that, although there were less reads mapping to sadA than to ubiquitous housekeeping genes (arcC and mutS), normalized reads counts were still >1000 times higher than those of rare control genes (icaA, icaB, and epiA). At protein sequence level SadA homologs were found in at least 7 phyla: Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, Chloroflexi, and Cyanobacteria, and in 23 genera of the phylum Firmicutes. A high proportion of the genera that have a SadA homolog belong to the classical skin and intestinal microbiota. The distribution of sadA in so many different phyla illustrates the importance of horizontal gene transfer (HGT). We show that the sadA gene is widely distributed in the human skin microbiome. When comparing the sadA read counts in the probands, there was no correlation between age and gender, but an enormous difference in the sadA read counts in the microbiome of the individuals. Since sadA is involved in TA synthesis, it is likely that the TA content of the skin is correlated with the amount of TA producing bacteria in the microbiome. In this way, the microbiome-generated TA could influence signal transmission in the epithelial and nervous system.

The Genome of Staphylococcus epidermidis O47.

The skin colonizing coagulase-negative Staphylococcus epidermidis causes nosocomial infections and is an important opportunistic and highly adaptable pathogen. To gain more insight into this species, we sequenced the genome of the biofilm positive, methicillin susceptible S. epidermidis O47 strain (hereafter O47). This strain belongs to the most frequently isolated sequence type 2. In comparison to the RP62A strain, O47 can be transformed, which makes it a preferred strain for molecular studies. S. epidermidis O47's genome has a single chromosome of about 2.5 million base pairs and no plasmid. Its oriC sequence has the same directionality as S. epidermidis RP62A, S. carnosus, S. haemolyticus, S. saprophyticus and is inverted in comparison to Staphylococcus aureus and S. epidermidis ATCC 12228. A phylogenetic analysis based on all S. epidermidis genomes currently available at GenBank revealed that O47 is closest related to DAR1907. The genome of O47 contains genes for the typical global regulatory systems known in staphylococci. In addition, it contains most of the genes encoding for the typical virulence factors for S. epidermidis but not for S. aureus with the exception of a putative hemolysin III. O47 has the typical S. epidermidis genetic islands and several mobile genetic elements, which include staphylococcal cassette chromosome (SCC) of about 54 kb length and two prophages φO47A and φO47B. However, its genome has no transposons and the smallest number of insertion sequence (IS) elements compared to the other known S. epidermidis genomes. By sequencing and analyzing the genome of O47, we provide the basis for its utilization in genetic and molecular studies of biofilm formation.

In Silico and in Vitro Study of Trace Amines (TA) and Dopamine (DOP) Interaction with Human Alpha 1-Adrenergic Receptor and the Bacterial Adrenergic Receptor QseC.

BACKGROUND/AIMS: Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and ß-adrenergic receptor (ß-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC. METHODS: We performed an in silico study using 3D structure from SWISS MODEL and analyzed the protein interaction via molecular docking using PyMol, PoseView and PyRX 8.0. For the in vitro study, we investigated the QseC kinase activity by measuring the remaining ATP in a reaction containing QseC-enriched membrane incubated together with purified QseB and EPI, TA, DOP, or PTL respectively. We also measured the intracellular Ca++ levels, which represents the α1-AR activation, in LNCAP (pancreatic cell line) cells treated with EPI, TA, DOP and PTL respectively using a fluorescence-based assay. The LNCAP cell proliferation was measured using an MTT-based assay. RESULTS: Our in silico analysis revealed that TAs and DOP have high binding affinity to the human α1-AR and the bacterial adrenergic receptor (QseC), comparable to epinephrine (EPI). Both are membrane-bound kinases. Experimental studies with pancreatic cell line (LNCAP) showed that the TAs and DOP act as α1-AR antagonist by counteracting the effect of EPI. In the presence of EPI, TA and DOP trigger an increase of the intracellular Ca++ levels in the LNCAP cells leading to an inhibition of cell proliferation. Although in silico data suggest an interaction of TA and DOP with QseC, they do not inhibit the kinase activity of QseC, a histidine kinase receptor involved in quorum sensing which is also sensitive to EPI. CONCLUSION: Our study showed that the TAs and DOP act as α1-AR antagonist but no effect was observed for QseC.

Trace amines produced by skin bacteria accelerate wound healing in mice.

Certain skin bacteria are able to convert aromatic amino acids (AAA) into trace amines (TA) that act as neuromodulators. Since the human skin and sweat contain a comparatively high content of AAA one can expect that such bacteria are able to produce TA on our skin. Here we show that TA-producing Staphylococcus epidermidis strains expressing SadA are predominant on human skin and that TA accelerate wound healing. In wounded skin, keratinocytes produce epinephrine (EPI) that leads to cell motility inhibition by ß2-adrenergic receptor (ß2-AR) activation thus delay wound healing. As ß2-AR antagonists, TA and dopamine (DOP) abrogate the effect of EPI thus accelerating wound healing both in vitro and in a mouse model. In the mouse model, the S. epidermidis wild type strain accelerates wound healing compared to its ΔsadA mutant. Our study demonstrates that TA-producing S. epidermidis strains present on our skin might be beneficial for wound healing.

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor.

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein-like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat-inducible and appears to play a major role in Lpl1 interaction. Pre-incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1-Hsp90 interaction induces F-actin formation, thus, triggering an endocytosis-like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl-Hsp90 interaction.

A new host cell internalisation pathway for SadA-expressing staphylococci triggered by excreted neurochemicals.

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface-bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal-pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface-bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT-29. We found that TAs and DOP are agonists of the α2-adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F-actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F-actin formation without decreasing the cytoplasmic cAMP level. The neurochemical-induced internalisation in host cells is independent of the fibronectin-binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

Genetic Adaptation of a Mevalonate Pathway Deficient Mutant in Staphylococcus aureus.

In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureusΔmvaS mutant can revert to prototrophy. This mutant couldn't grow in the absence of MVA. However, after a long lag-phase of 4-6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ΔmvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ΔmvaS mutant.

Toll-Like Receptor 2 and Lipoprotein-Like Lipoproteins Enhance Staphylococcus aureus Invasion in Epithelial Cells.

Staphylococcus aureus contains a certain subclass of lipoproteins, the so-called lipoprotein-like lipoproteins (Lpl's), that not only represent Toll-like receptor 2 (TLR2) ligands but are also involved in host cell invasion. Here we addressed the question of which factors contribute to Lpl-mediated invasion of epithelial cells and keratinocytes. For this purpose, we compared the invasiveness of USA300 and its Δlpl mutant under different conditions. In the presence of the matrix proteins IgG, fibrinogen (Fg), and fibronectin (Fn), and of fetal bovine serum (FBS), the invasion ratio was increased in both strains, and always more in USA300 than in its Δlpl mutant. Interestingly, when we compared the invasion of HEK-0 and HEK-TLR2 cells, the cells expressing TLR2 showed a 9-times-higher invasion frequency. When HEK-TLR2 cells were additionally stimulated with a synthetic lipopeptide, Pam3CSK4 (P3C), the invasion frequency was further increased. A potential reason for the positive effect of TLR2 on invasion could be that TLR2 activation by P3C also activates F-actin formation. Here we show that S. aureus invasion depends on a number of factors, on the host side as well as on the bacterial side.

Lantibiotic production is a burden for the producing staphylococci.

Lantibiotics are antimicrobial peptides that contain non-proteinogenic amino acids lanthionine and 3-methyllanthionine and are produced by Gram-positive bacteria. Here we addressed the pros and cons of lantibiotic production for its producing strains. Two staphylococcal strains, S. gallinarum Tü3928 and S. epidermidis Tü3298 producing gallidermin and epidermin respectively were selected. In each of these parental strains, the structural genes gdmA and epiA were deleted; all the other biosynthetic genes including the immunity genes were left intact. Comparative analysis of the lantibiotic-producing strains with their non-producing mutants revealed that lantibiotic production is a burden for the cells. The production affected growth, caused release of ATP, lipids and increased the excretion of cytoplasmic proteins (ECP). The epidermin and gallidermin immunity genes were insufficient to protect the cells from their own product. Co-cultivation studies showed that the ΔgdmA mutant has an advantage over the parental strain; the latter was outcompeted. On the one hand, the production of staphylococcal lantibiotics is beneficial by suppressing competitors, but on the other hand they impose a burden on the producing-strains when they accumulate in higher amounts. Our observations explain why antibiotic-producing strains occur as a minority on our skin and other ecological niches, but retain corresponding antibiotic resistance.

SadA-Expressing Staphylococci in the Human Gut Show Increased Cell Adherence and Internalization.

A subgroup of biogenic amines, the so-called trace amines (TAs), are produced by mammals and bacteria and can act as neuromodulators. In the genus Staphylococcus, certain species are capable of producing TAs through the activity of staphylococcal aromatic amino acid decarboxylase (SadA). SadA decarboxylates aromatic amino acids to produce TAs, as well as dihydroxy phenylalanine and 5-hydroxytryptophan to thus produce the neurotransmitters dopamine and serotonin. SadA-expressing staphylococci were prevalent in the gut of most probands, where they are part of the human intestinal microflora. Furthermore, sadA-expressing staphylococci showed increased adherence to HT-29 cells and 2- to 3-fold increased internalization. Internalization and adherence was also increased in a sadA mutant in the presence of tryptamine. The α2-adrenergic receptor is required for enhanced adherence and internalization. Thus, staphylococci in the gut might contribute to gut activity and intestinal colonization.

Staphylococcal (phospho)lipases promote biofilm formation and host cell invasion.

Most Staphylococcus aureus strains secrete two lipases SAL1 and SAL2 encoded by gehA and gehB. These two lipases differ with respect to their substrate specificity. Staphylococcus hyicus secretes another lipase, SHL, which is in contrast to S. aureus lipases Ca2+-dependent and has a broad-spectrum lipase and phospholipase activity. The aim of this study was to investigate the role of staphylococcal (phospho) lipases in virulence. For this we constructed a gehA-gehB double deletion mutant in S. aureus USA300 and expressed SHL in agr-positive (accessory gene regulator) and agr-negative S. aureus strains. The lipases themselves have no hemolytic or cytotoxic activity. However, in agr-negative strains SHL-expression caused an upregulation of hemolytic activity. We further show that SHL-expression significantly enhanced biofilm formation probably due to an increase of extracellular DNA release. SHL-expression also increased host cell invasion 4-6-fold. Trioleate, a main triacylglycerol component of mammalian skin, induced lipase production. Finally, in the mouse sepsis and skin colonization models the lipase producing and mutant strain showed no significant difference compared to the WT strain. In conclusion, we show that staphylococcal lipases promote biofilm formation and host cell invasion and thereby contribute to S. aureus virulence.