Effect of TraN key residues involved in DNA binding on pIP501 transfer rates in Enterococcus faecalis.

Conjugation is a major mechanism that facilitates the exchange of antibiotic resistance genes among bacteria. The broad-host-range Inc18 plasmid pIP501 harbors 15 genes that encode for a type IV secretion system (T4SS). It is a membrane-spanning multiprotein complex formed between conjugating donor and recipient cells. The penultimate gene of the pIP501 operon encodes for the cytosolic monomeric protein TraN. This acts as a transcriptional regulator by binding upstream of the operon promotor, partially overlapping with the origin of transfer. Additionally, TraN regulates traN and traO expression by binding upstream of the PtraNO promoter. This study investigates the impact of nine TraN amino acids involved in binding to pIP501 DNA through site-directed mutagenesis by exchanging one to three residues by alanine. For three traN variants, complementation of the pIP501∆traN knockout resulted in an increase of the transfer rate by more than 1.5 orders of magnitude compared to complementation of the mutant with native traN. Microscale thermophoresis (MST) was used to assess the binding affinities of three TraN double-substituted variants and one triple-substituted variant to its cognate pIP501 double-stranded DNA. The MST data strongly correlated with the transfer rates obtained by biparental mating assays in Enterococcus faecalis. The TraN variants TraN_R23A-N24A-Q28A, TraN_H82A-R86A, and TraN_G100A-K101A not only exhibited significantly lower DNA binding affinities but also, upon complementation of the pIP501∆traN knockout, resulted in the highest pIP501 transfer rates. This confirms the important role of the TraN residues R23, N24, Q28, H82, R86, G100, and K101 in downregulating pIP501 transfer. Although TraN is not part of the mating pair formation complex, TraE, TraF, TraH, TraJ, TraK, and TraM were coeluted with TraN in a pull-down. Moreover, TraN homologs are present not only in Inc18 plasmids but also in RepA_N and Rep_3 family plasmids, which are frequently found in enterococci, streptococci, and staphylococci. This points to a widespread role of this repressor in conjugative plasmid transfer among Firmicutes.

Special Issue "Multidrug-Resistant Bacteria in the Environment, Their Resistance and Transfer Mechanisms".

Multidrug-resistant bacteria are an emerging issue which is not restricted to clinics and the health care sector, but is increasingly affecting the environment [...].

Fate of Horizontal-Gene-Transfer Markers and Beta-Lactamase Genes during Thermophilic Composting of Human Excreta.

Thermophilic composting is a suitable treatment for the recycling of organic wastes for agriculture. However, using human excreta as feedstock for composting raises concerns about antibiotic resistances. We analyzed samples from the start and end of a thermophilic composting trial of human excreta, together with green cuttings and straw, with and without biochar. Beta-lactamase genes blaCTX-M, blaIMP, and blaTEM conferring resistance to broad-spectrum beta-lactam antibiotics, as well as horizontal gene transfer marker genes, intI1 and korB, were quantified using qPCR. We found low concentrations of the beta-lactamase genes in all samples, with non-significant mean decreases in blaCTX-M and blaTEM copy numbers and a mean increase in blaIMP copy numbers. The decrease in both intI1 and korB genes from start to end of composting indicated that thermophilic composting can decrease the horizontal spread of resistance genes. Thus, thermophilic composting can be a suitable treatment for the recycling of human excreta.

Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms.

Most bacteria attach to biotic or abiotic surfaces and are embedded in a complex matrix which is known as biofilm. Biofilm formation is especially worrisome in clinical settings as it hinders the treatment of infections with antibiotics due to the facilitated acquisition of antibiotic resistance genes (ARGs). Environmental settings are now considered as pivotal for driving biofilm formation, biofilm-mediated antibiotic resistance development and dissemination. Several studies have demonstrated that environmental biofilms can be hotspots for the dissemination of ARGs. These genes can be encoded on mobile genetic elements (MGEs) such as conjugative and mobilizable plasmids or integrative and conjugative elements (ICEs). ARGs can be rapidly transferred through horizontal gene transfer (HGT) which has been shown to occur more frequently in biofilms than in planktonic cultures. Biofilm models are promising tools to mimic natural biofilms to study the dissemination of ARGs via HGT. This review summarizes the state-of-the-art of biofilm studies and the techniques that visualize the three main HGT mechanisms in biofilms: transformation, transduction, and conjugation.

Degradation of alkane hydrocarbons by Priestia megaterium ZS16 and sediments consortia with special reference to toxicity and oxidative stress induced by the sediments in the vicinity of an oil refinery.

Petroleum hydrocarbon is a critical ecological issue with impact on ecosystems through bioaccumulation. It poses significant risks to human health. Due to the extent of alkane hydrocarbon pollution in some environments, biosurfactants are considered as a new multifunctional technology for the efficient removal of petroleum-based contaminants. To this end, Yamuna river sediments were collected at different sites in the vicinity of Mathura oil refinery, UP (India). They were analysed by atomic absorption spectrophotometry and gas chromatography-mass spectrometry (GC-MS) for heavy metals and organic pollutants. Heptadecane, nonadecane, oleic acid ester and phthalic acid were detected. In total 107 bacteria were isolated from the sediments and screened for biosurfactant production. The most efficient biosurfactant producing strain was tested for its capability to degrade hexadecane efficiently at different time intervals (0 h, 7 d, 14 d and 21 d). FT-IR analysis defined the biosurfactant as lipopeptide. 16S rRNA gene sequencing identified the bacterium as Priestia megaterium. The strain lacks resistance to common antibiotics thus making it an important candidate for remediation. The microbial consortia present in the sediments were also investigated for their capability to degrade C16, C17 and C18 alkane hydrocarbons. By using gas chromatography-mass spectrophotometry the metabolites were identified as 1-docosanol, dodecanoic acid, 7-hexadecenal, (Z)-, hexadecanoic acid, docosanoic acid, 1-hexacosanal, 9-octadecenoic acid, 3-octanone, Z,Z-6,28-heptatriactontadien-2-one, heptacosyl pentafluoropropionate, 1,30-triacontanediol and decyl octadecyl ester. Oxidative stress in Vigna radiata L. roots was observed by using Confocal Laser Scanning Microscopy. A strong reduction in seed germination and radicle and plumule length was observed when Vigna radiata L. was treated with different concentrations of sediment extracts, possibly due to the toxic effects of the pollutants in the river sediments. Thus, this study is significant since it considers the toxicological effects of hydrocarbons and to degrade them in an environmentally friendly manner.

Small Things Matter: The 11.6-kDa TraB Protein is Crucial for Antibiotic Resistance Transfer Among Enterococci.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci. In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely.

Metagenomic Insights Into the Changes of Antibiotic Resistance and Pathogenicity Factor Pools Upon Thermophilic Composting of Human Excreta.

In times of climate change, practicing a form of sustainable, climate-resilient and productive agriculture is of primordial importance. Compost could be one form of sustainable fertilizer, which is increasing humus, water holding capacity, and nutrient contents of soils. It could thereby strengthen agriculture toward the adverse effects of climate change, especially when additionally combined with biochar. To get access to sufficient amounts of suitable materials for composting, resources, which are currently treated as waste, such as human excreta, could be a promising option. However, the safety of the produced compost regarding human pathogens, pharmaceuticals (like antibiotics) and related resistance genes must be considered. In this context, we have investigated the effect of 140- and 154-days of thermophilic composting on the hygienization of human excreta and saw dust from dry toilets together with straw and green cuttings with and without addition of biochar. Compost samples were taken at the beginning and end of the composting process and metagenomic analysis was conducted to assess the fate of antibiotic resistance genes (ARGs) and pathogenicity factors of the microbial community over composting. Potential ARGs conferring resistance to major classes of antibiotics, such as beta-lactam antibiotics, vancomycin, the MLSB group, aminoglycosides, tetracyclines and quinolones were detected in all samples. However, relative abundance of ARGs decreased from the beginning to the end of composting. This trend was also found for genes encoding type III, type IV, and type VI secretion systems, that are involved in pathogenicity, protein effector transport into eukaryotic cells and horizontal gene transfer between bacteria, respectively. The results suggest that the occurrence of potentially pathogenic microorganisms harboring ARGs declines during thermophilic composting. Nevertheless, ARG levels did not decline below the detection limit of quantitative PCR (qPCR). Thresholds for the usage of compost regarding acceptable resistance gene levels are yet to be evaluated and defined.

Thermophilic Composting of Human Feces: Development of Bacterial Community Composition and Antimicrobial Resistance Gene Pool.

In times of climate change, practicing sustainable, climate-resilient, and productive agriculture is of primordial importance. Compost from different resources, now treated as wastes, could be one form of sustainable fertilizer creating a resilience of agriculture to the adverse effects of climate change. However, the safety of the produced compost regarding human pathogens, pharmaceuticals, and related resistance genes must be considered. We have assessed the effect of thermophilic composting of dry toilet contents, green cuttings, and straw, with and without biochar, on fecal indicators, the bacterial community, and antibiotic resistance genes (ARGs). Mature compost samples were analyzed regarding fecal indicator organisms, revealing low levels of Escherichia coli that are in line with German regulations for fertilizers. However, one finding of Salmonella spp. exceeded the threshold value. Cultivation of bacteria from the mature compost resulted in 200 isolates with 36.5% of biosafety level 2 (BSL-2) species. The majority is known as opportunistic pathogens that likewise occur in different environments. A quarter of the isolated BSL-2 strains exhibited multiresistance to different classes of antibiotics. Molecular analysis of total DNA before and after composting revealed changes in bacterial community composition and ARGs. 16S rRNA gene amplicon sequencing showed a decline of the two most abundant phyla Proteobacteria (start: 36-48%, end: 27-30%) and Firmicutes (start: 13-33%, end: 12-16%), whereas the abundance of Chloroflexi, Gemmatimonadetes, and Planctomycetes rose. Groups containing many human pathogens decreased during composting, like Pseudomonadales, Bacilli with Bacillus spp., or Staphylococcaceae and Enterococcaceae. Gene-specific PCR showed a decline in the number of detectable ARGs from 15 before to 8 after composting. The results reveal the importance of sufficiently high temperatures lasting for a sufficiently long period during the thermophilic phase of composting for reducing Salmonella to levels matching the criteria for fertilizers. However, most severe human pathogens that were targeted by isolation conditions were not detected. Cultivation-independent analyses also indicated a decline in bacterial orders comprising many pathogenic bacteria, as well as a decrease in ARGs. In summary, thermophilic composting could be a promising approach for producing hygienically safe organic fertilizer from ecological sanitation.

Conjugation Operons in Gram-Positive Bacteria with and without Antitermination Systems.

Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription undermines this strict regulation, particularly affecting large operons. The negative effects of spurious transcription can be mitigated by the presence of multiple terminators inside the operon, in combination with an antitermination system. Antitermination systems modify the transcription elongation complexes and enable them to bypass terminators. Bacterial conjugation is the process by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugation involves many genes that are mostly organized in one or a few large operons. It has recently been shown that many conjugation operons present on plasmids replicating in Gram-positive bacteria possess a bipartite antitermination system that allows not only many terminators inside the conjugation operon to be bypassed, but also the differential expression of a subset of genes. Here, we show that some conjugation operons on plasmids belonging to the Inc18 family of Gram-positive broad host-range plasmids do not possess an antitermination system, suggesting that the absence of an antitermination system may have advantages. The possible (dis)advantages of conjugation operons possessing (or not) an antitermination system are discussed.

Antimicrobials Functioning through ROS-Mediated Mechanisms: Current Insights.

Antibiotic resistance and infections caused by multidrug-resistant bacteria are global health concerns. Reducing the overuse and misuse of antibiotics is the primary step toward minimizing the antibiotic resistance crisis. Thus, it is imperative to introduce and implement novel antimicrobial strategies. Recently, several alternative antimicrobials targeting oxidative stress in bacteria have been studied and shown to be promising. Oxidative stress occurs when bacterial cells fail to detoxify the excessive reactive oxygen species (ROS) accumulated in the cells. Bacteria deploy numerous defense mechanisms against oxidative stress. The oxidative stress response is not essential for the normal growth of bacteria, but it is crucial for their survival. This toxic oxidative stress is created by the host immune response or antimicrobials generating ROS. ROS possess strong oxidation potential and cause serious damage to nucleic acids, lipids, and proteins. Since ROS-based antimicrobials target multiple sites in bacteria, these antimicrobials have attracted the attention of several researchers. In this review, we present recent ROS-based alternative antimicrobials and strategies targeting oxidative stress which might help in mitigating the problem of antibiotic resistance and dissemination.

Transcriptomic analysis of stress response to novel antimicrobial coatings in a clinical MRSA strain.

Multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause nosocomial infections that can have deleterious effects on human health. Thus, it is imperative to find solutions to treat these detrimental infections as well as to control their spread. We tested the effect of two different antimicrobial materials, functionalised graphene oxide (GOX), and AGXX® coated on cellulose fibres, on the growth and transcriptome of the clinical MRSA strain S. aureus 04-02981. In addition, we investigated the effect of a third material as a combination of GOX and AGXX® fibres on S. aureus 04-02981. Standard plate count assay revealed that the combination of fibres, GOX-AGXX® inhibited the growth of S. aureus 04-02981 by 99.98%. To assess the effect of these antimicrobials on the transcriptome of our strain, cultures of S. aureus 04-02981 were incubated with GOX, AGXX®, or GOX-AGXX® fibres for different time periods and then subjected to RNA-sequencing. Uncoated cellulose fibres were used as a negative control. The antimicrobial fibres had a huge impact on the transcriptome of S. aureus 04-02981 affecting the expression of 2650 genes. Primarily genes related to biofilm formation and virulence (such as agr, sarA, and those of the two-component system SaeRS), and genes crucial for survival in biofilms (like arginine metabolism arc genes) were repressed. In contrast, the expression of siderophore biosynthesis genes (sbn) was induced, a probable response to stress imposed by the antimicrobials and the conditions of iron-deficiency. Genes associated with potassium transport, intracellular survival and pathogenesis (kdp) were also differentially expressed. Our data suggest that the combination of GOX and AGXX® acts as an efficient antimicrobial against S. aureus 04-02981. Thus, these materials are potential candidates for applications in antimicrobial surface coatings.

Novel Antimicrobial Cellulose Fleece Inhibits Growth of Human-Derived Biofilm-Forming Staphylococci During the SIRIUS19 Simulated Space Mission.

Two novel antimicrobial surface coatings were assessed for their lasting antibacterial effect under simulated space conditions during the SIRIUS-19 study. Because long-term space travel can affect the human immune system, astronauts are particularly susceptible to infectious disease. Moreover, the space flight environment can alter the composition of microbial communities within the spacecraft and increase bacterial virulence and resistance to antibiotics. In addition to protecting the crew from infection by human pathogens, prevention and elimination of bacterial contamination is important to avoid corrosion and damage of the technical equipment. The antimicrobial coating AGXX® consists of micro-galvanic cells composed of silver and ruthenium which damage bacterial cells through the release of reactive oxygen species. Over the last years, several studies on the antimicrobial effect of AGXX® have demonstrated an effective inhibition of growth and even complete elimination of many pathogenic bacteria - including multiresistant microorganisms - as well as their biofilms. The second antimicrobial coating, GOX, consists of chemically modified graphene oxide. Through a positive surface charge and its flexible scaffold, GOX can multivalently bind and immobilize bacteria via electrostatic attraction. Here, AGXX® and GOX were applied to non-metallic carriers not previously tested. The antimicrobial coated materials, as well as uncoated control samples, were exposed in the SIRIUS artificial space module and analyzed at different time points during the 4-months isolation study. Survival and growth of airborne heterotrophic, aerobic bacteria on the surfaces were assessed by cultivation-based methods, employing growth conditions suitable for potential human pathogens. Human-associated, biofilm-forming Staphylococcus spp. (S. hominis, S. haemolyticus, and S. epidermidis) strongly dominated at all time points, most were resistant against erythromycin, kanamycin, and ampicillin. AGXX® coatings completely inhibited growth of these opportunistic pathogens on all tested surface materials. Particularly, AGXX®-cellulose fleece achieved a clear reduction in bacterial load able to recover post contact. GOX-cellulose fleece effectively immobilized bacteria. Sequence analysis of 16S rRNA gene amplicons revealed that the isolated Staphylococcus spp. did not dominate the overall bacterial community, accounting for only 0.1-0.4% of all sequences. Instead, molecular data revealed Lactobacillus, Comamonas, Pseudomonas, Sporosarcina, and Bacillus as the dominant genera across all samples and time points.

Multi-resistant biofilm-forming pathogens on the International Space Station.

The International Space Station (ISS) is a confined and closed habitat with unique conditions such as cosmic radiation, and microgravity. These conditions have a strong effect on the human and spacecraft microflora. They can affect the immune response of the crew-members, thus posing a threat to their health. Microbial diversity and abundance of microorganisms from surfaces, air filters and air samples on the ISS have been studied. Enterobacteriaceae, Bacillus spp., Propionibacterium spp., Corynebacterium spp., and Staphylococcus spp. were among the most frequently isolated bacteria. Microbial growth, biofilm formation, stress response, and pathogenicity are affected by microgravity. Increased resistance to antibiotics in bacteria isolated from the ISS has often been reported. Enterococcus faecalis and Staphylococcus spp. isolates from the ISS have been shown to harbor plasmid-encoded transfer genes. These genes facilitate the dissemination of antibiotic resistances. These features of ISS-pathogens call for novel approaches including highly effective antimicrobials which can be easily used on the ISS. A promising material is the antimicrobial surface coating AGXX, a self-recycling material consisting of two noble metals. It drastically reduced microbial growth of multi-resistant human pathogens, such as staphylococci and enterococci. Further novel approaches include the application of cold atmospheric plasma for the sterilization of spacecrafts.

Regulation of Gram-Positive Conjugation.

Type IV Secretion Systems (T4SSs) are membrane-spanning multiprotein complexes dedicated to protein secretion or conjugative DNA transport (conjugation systems) in bacteria. The prototype and best-characterized T4SS is that of the Gram-negative soil bacterium Agrobacterium tumefaciens. For Gram-positive bacteria, only conjugative T4SSs have been characterized in some biochemical, structural, and mechanistic details. These conjugation systems are predominantly encoded by self-transmissible plasmids but are also increasingly detected on integrative and conjugative elements (ICEs) and transposons. Here, we report regulatory details of conjugation systems from Enterococcus model plasmids pIP501 and pCF10, Bacillus plasmid pLS1, Clostridium plasmid pCW3, and staphylococcal plasmid pSK41. In addition, regulation of conjugative processes of ICEs (ICEBs1, ICESt1, ICESt3) by master regulators belonging to diverse repressor families will be discussed. A special focus of this review lies on the comparison of regulatory mechanisms executed by proteins belonging to the RRNPP family. These regulators share a common fold and govern several essential bacterial processes, including conjugative transfer.

Biofilm Forming Antibiotic Resistant Gram-Positive Pathogens Isolated From Surfaces on the International Space Station.

The International Space Station (ISS) is a closed habitat in a uniquely extreme and hostile environment. Due to these special conditions, the human microflora can undergo unusual changes and may represent health risks for the crew. To address this problem, we investigated the antimicrobial activity of AGXX®, a novel surface coating consisting of micro-galvanic elements of silver and ruthenium along with examining the activity of a conventional silver coating. The antimicrobial materials were exposed on the ISS for 6, 12, and 19 months each at a place frequently visited by the crew. Bacteria that survived on the antimicrobial coatings [AGXX® and silver (Ag)] or the uncoated stainless steel carrier (V2A, control material) were recovered, phylogenetically affiliated and characterized in terms of antibiotic resistance (phenotype and genotype), plasmid content, biofilm formation capacity and antibiotic resistance transferability. On all three materials, surviving bacteria were dominated by Gram-positive bacteria and among those by Staphylococcus, Bacillus and Enterococcus spp. The novel antimicrobial surface coating proved to be highly effective. The conventional Ag coating showed only little antimicrobial activity. Microbial diversity increased with increasing exposure time on all three materials. The number of recovered bacteria decreased significantly from V2A to V2A-Ag to AGXX®. After 6 months exposure on the ISS no bacteria were recovered from AGXX®, after 12 months nine and after 19 months three isolates were obtained. Most Gram-positive pathogenic isolates were multidrug resistant (resistant to more than three antibiotics). Sulfamethoxazole, erythromycin and ampicillin resistance were most prevalent. An Enterococcus faecalis strain recovered from V2A steel after 12 months exposure exhibited the highest number of resistances (n = 9). The most prevalent resistance genes were ermC (erythromycin resistance) and tetK (tetracycline resistance). Average transfer frequency of erythromycin, tetracycline and gentamicin resistance from selected ISS isolates was 10-5 transconjugants/recipient. Most importantly, no serious human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) were found on any surface. Thus, the infection risk for the crew is low, especially when antimicrobial surfaces such as AGXX® are applied to surfaces prone to microbial contamination.

TraN: A novel repressor of an Enterococcus conjugative type IV secretion system.

The dissemination of multi-resistant bacteria represents an enormous burden on modern healthcare. Plasmid-borne conjugative transfer is the most prevalent mechanism, requiring a type IV secretion system that enables bacteria to spread beneficial traits, such as resistance to last-line antibiotics, among different genera. Inc18 plasmids, like the Gram-positive broad host-range plasmid pIP501, are substantially involved in propagation of vancomycin resistance from Enterococci to methicillin-resistant strains of Staphylococcus aureus. Here, we identified the small cytosolic protein TraN as a repressor of the pIP501-encoded conjugative transfer system, since deletion of traN resulted in upregulation of transfer factors, leading to highly enhanced conjugative transfer. Furthermore, we report the complex structure of TraN with DNA and define the exact sequence of its binding motif. Targeting this protein-DNA interaction might represent a novel therapeutic approach against the spreading of antibiotic resistances.

Enterococcus adhesin PrgB facilitates type IV secretion by condensation of extracellular DNA.

Conjugative type IV secretion systems (T4SSs) are multi-protein complexes in Gram-negative and Gram-positive (G+) bacteria, responsible for spreading antibiotic resistances and virulence factors among different species. Compared to Gram-negative bacteria, which establish close contacts for conjugative transfer via sex pili, G+ T4SSs are suggested to employ surface adhesins instead. One example is pCF10, an enterococcal conjugative sex-pheromone responsive plasmid with a narrow host range, thus disseminating genetic information only among closely related species. This MicroCommentary is dedicated to the crystal structure of the pCF10-encoded adhesion domain of PrgB presented by Schmitt et al. The authors show in their work that this adhesion domain is responsible for biofilm formation, tight binding and condensation of extracellular DNA (eDNA) and conjugative transfer of pCF10. A sophisticated two-step mechanism for highly efficient conjugative transfer is postulated, including the formation of PrgB-mediated long-range intercellular contacts by binding and establishment of shorter-range contacts via condensation of eDNA. PrgB binding to lipoteichoic acid on the recipient cell surface stabilizes junctions between the mating partners. The major findings by Schmitt et al. will be brought into a broader context and potential medical applications targeting eDNA as essential component in biofilm formation and conjugation will be discussed.

Broad-host-range Inc18 plasmids: Occurrence, spread and transfer mechanisms.

Conjugative plasmid transfer is one of the major mechanisms responsible for the spread of antibiotic resistance and virulence genes. The incompatibility (Inc) 18 group of plasmids is a family of plasmids replicating by the theta-mechanism, whose members have been detected frequently in enterococci and streptococci. Inc18 plasmids encode a variety of antibiotic resistances, including resistance to vancomycin, chloramphenicol and the macrolide-lincosamide-streptogramine (MLS) group of antibiotics. These plasmids comprising insertions of Tn1546 were demonstrated to be responsible for the transfer of vancomycin resistance encoded by the vanA gene from vancomycin resistant enterococci (VRE) to methicillin resistant Staphylococcus aureus (MRSA). Thereby vancomycin resistant S. aureus (VRSA) were generated, which are serious multi-resistant pathogens challenging the health care system. Inc18 plasmids are widespread in the clinic and frequently have been detected in the environment, especially in domestic animals and wastewater. pIP501 is one of the best-characterized conjugative Inc18 plasmids. It was originally isolated from a clinical Streptococcus agalactiae strain and is, due to its small size and simplicity, a model to study conjugative plasmid transfer in Gram-positive bacteria. Here, we report on the occurrence and spread of Inc18-type plasmids in the clinic and in different environments as well as on the exchange of the plasmids among them. In addition, we discuss molecular details on the transfer mechanism of Inc18 plasmids and its regulation, as exemplified by the model plasmid pIP501. We finish with an outlook on promising approaches on how to reduce the emerging spread of antibiotic resistances.

A Novel Antimicrobial Coating Represses Biofilm and Virulence-Related Genes in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important cause of hospital-acquired infections worldwide. It is one of the most threatening pathogens due to its multi-drug resistance and strong biofilm-forming capacity. Thus, there is an urgent need for novel alternative strategies to combat bacterial infections. Recently, we demonstrated that a novel antimicrobial surface coating, AGXX®, consisting of micro-galvanic elements of the two noble metals, silver and ruthenium, surface-conditioned with ascorbic acid, efficiently inhibits MRSA growth. In this study, we demonstrated that the antimicrobial coating caused a significant reduction in biofilm formation (46%) of the clinical MRSA isolate, S. aureus 04-02981. To understand the molecular mechanism of the antimicrobial coating, we exposed S. aureus 04-02981 for different time-periods to the coating and investigated its molecular response via next-generation RNA-sequencing. A conventional antimicrobial silver coating served as a control. RNA-sequencing demonstrated down-regulation of many biofilm-associated genes and of genes related to virulence of S. aureus. The antimicrobial substance also down-regulated the two-component quorum-sensing system agr suggesting that it might interfere with quorum-sensing while diminishing biofilm formation in S. aureus 04-02981.