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.

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.

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.

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.

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.

Stress response of a clinical Enterococcus faecalis isolate subjected to a novel antimicrobial surface coating.

Emerging antibiotic resistance among pathogenic bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem for effective and long-term decontamination in health-care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the impact of AGXX®, a novel broad-spectrum antimicrobial surface coating consisting of micro-galvanic elements formed by silver and ruthenium, on the transcriptome of Enterococcus faecalis. A clinical E. faecalis isolate was subjected to metal stress by growing it for different periods in presence of the antimicrobial coating or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression in presence of the antimicrobial materials with focus on known stress genes. Exposure to the antimicrobial coating had a large impact on the transcriptome of E. faecalis. After 24min almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to the antimicrobial coating induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES and the Clp proteases, ClpE and ClpB, were among the top up-regulated heat shock genes. Differential expression of thioredoxin, superoxide dismutase and glutathione synthetase genes indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXX® results in a synergistic antimicrobial effect, superior to that of conventional silver coatings.

Mossambicus tilapia (Oreochromis mossambicus) collected from water bodies impacted by urban waste carries extended-spectrum beta-lactamases and integron-bearing gut bacteria.

Oreochromis mossambicus (Peters 1852) (Tilapia) is one of the most consumed fish globally. Tilapia thrives well in environments polluted by urban waste, which invariably contain antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Thus, Tilapia surviving in such polluted environments may serve as a potential source for dissemination of ARGs. To investigate this, we isolated bacterial strains from gut of Tilapia found in polluted rivers and lakes near Pune, India, and studied the prevalence of resistance genes by molecular methods. A total of 91 bacterial strains were obtained, which include fish pathogens and human pathogens such as Aeromonas hydrophila, Klebsiella pneumoniae, E. coli, Serratia marcescens, Enterobacter spp. and Shigella spp. Overall the prevalence of class 1 integrons, class 2 integrons, extended-spectrum betalactamases (ESBLs) blaCTX-M, blaSHV and aac(6')-Ib-cr gene was 38 percent, 24 percent, 38 percent, 31 percent and 31 percent respectively. Forty-two percent of the Enterobacteriaceae strains carried blaCTX-M gene, which is a common ESBL gene in clinics. The study demonstrates that tilapia found in the polluted waters can serve as reservoirs and an alternative route for human exposure to clinically important ARG-carrying bacteria. The consumption and handling of these fish may pose a potential health risk.

Pantoea intestinalis sp. nov., isolated from the human gut.

A novel bacterial strain, 29Y89BT, was isolated from a faecal sample of a healthy human subject. Cells were Gram-stain-negative, motile, non-spore-forming and rod-shaped. Strain 29Y89BT formed cream-coloured colonies 2 mm in diameter on trypticase soy agar and showed optimum growth at 35 °C. Strain 29Y89BT showed highest 16S rRNA gene sequence similarity to Pantoea gaviniae A18/07T (98.4 %) followed by Pantoea calida 1400/07T (97.2 %). Multi-locus sequence analysis using atpD (ATP synthase ß subunit), gyrB (DNA gyrase), infB (initiation translation factor 2) and rpoB (RNA polymerase ß subunit) genes also supported the result of 16S rRNA gene sequence based phylogeny. Strain 29Y89BT showed 62 and 40.7 % DNA-DNA relatedness with P. calida DSM 22759T and P. gaviniae DSM 22758T. Strain 29Y89BT contained C17 : 0 cyclo, C19 : 0 cyclo ω8c, C16 : 0, C14 : 0 and C12 : 0 as predominant fatty acids. In addition, strain 29Y89BT showed physiological and phenotypic differences from its closest relatives P. gaviniae DSM 22758T and P. calida DSM 22759T. The polar lipid profile mainly comprised phospholipids. The DNA G+C content was 59.1 mol%. Thus, based on the findings of the current study, strain 29Y89BT showed clear delineations from its closest relatives P. gaviniae DSM 22758T and P. calida DSM 22759T, and is thus considered to represent a novel species of the genus Pantoea, for which the name Pantoea intestinalis sp. nov. is proposed. The type strain is 29Y89BT ( = DSM 28113T = MCC 2554T).