Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen. Vibrio vulnificus is a natural inhabitant of estuarine and other coastal marine environments where it exists free living or becomes concentrated in filter-feeding molluscan shellfish, such as oysters. The bacterium gains entrance to the human host through consumption of contaminated water or food, particularly raw or undercooked oysters and other seafood. Alternatively, infection occurs through open wounds that contact seawater or shellfish containing the bacterium [V. vulnificus notoriously causes an acute fulminating septicemia as well as severe wound infections. A third illness, gastroenteritis, is not life threatening and is typically not reported. Septicemic patients succumb to death in as little as 24 hours and in over 50% of cases. V. vulnificus is responsible for the vast majority of reported deaths associated with seafood consumption in the U.S. [acterium . V. vulnthe U.S. . Patientthe U.S. ,4. V. vulnificus, including its acid resistance, as well as its production of capsular polysaccharide (CPS), lipopolysaccharide (LPS), iron acquisition systems, cytotoxic factors, and motility and adherence/adhesion molecules [V. vulnificus CPS structure, function, and genetics, as well as control of CPS production via phase variation and other known environmental, genetic, and regulatory factors. A number of factors contribute to virulence of olecules . Unlike olecules ,5. Here,V. vulnificus was due, at least in part, to an antiphagocytic surface molecule [V. vulnificus strains, namely FCC and ATCC 27562  first presented evidence that virulence of molecule . In a fomolecule . Ruthenimolecule . Amako eCC 27562 , were di1 first pV. vulnificus strains was similarly characterized [V. vulnificus). The presence of capsular material again correlated with increased virulence in mice and with resistance to NHS, as well as with antiphagocytic ability, and increased invasiveness in subcutaneous tissue of guinea pigs [V. vulnificus was apparent. Separately, variation in capsule production among four other cterized . Opaque nea pigs . In a monea pigs . Thus, aV. vulnificus in other studies as well [This relationship was subsequently explored further using transposon mutagenesis. Compared to the opaque clinical isolate MO6-24/O , two tra as well ,13,14.V. vulnificus CPS was first reported for the clinical isolate MO6-24/O following extensive purification of its capsular material and analysis by nuclear magnetic resonance (NMR) spectroscopy. The MO6-24/O CPS polymer consists of four-sugar repeat subunits, each containing three residues of 2-acetamido-2,6-dideoxyhexopyranose in the α-gluco configuration ) and one residue of 2-acetamido hexuronate in the α-galactopyranose configuration ) [N-acetyl muramic acid, a well-known component of peptidoglycan, as well as evidence that the four-sugar repeat subunits for this strain are linked to serine via an amide bond [Carbohydrate composition of alNAcA)) . FurtheralNAcA)) . SimilaralNAcA)) . Analysiide bond . V. vulnificus CPS was poorly immunogenic in mice and rabbits [V. vulnificus CPS, which involved HPAEC analysis coupled with electrochemical detection and computer automation. Analysis of CPS from 120 V. vulnificus clinical and environmental isolates revealed that they could be divided into 94 ‘carbotypes’, each differing in their carbohydrate composition [Purified  rabbits . However rabbits , while a rabbits . In the  rabbits . Such prV. vulnificus. Moreover, specific virulence-related functions, such as antiphagocytic ability and resistance to the bacteriocidal action of human serum, appeared to be associated with CPS. Kreger et al. 1981 reported that a virulent strain of V. vulnificus was more resistant to phagocytosis by human polymorphonuclear (PMN) leukocytes than an attenuated strain, and their evidence suggested the possible involvement of an antiphagocytic antigen on the surface of the virulent isolate, which was later identified as an acidic polysaccharide [V. vulnificus also resisted phagocytosis by murine peritoneal macrophages in the absence of serum opsonins, a result which reinforced the notion that the surface of virulent cells  had antiphagocytic properties [As described earlier, studies conducted in the early to mid-1980s firmly established a correlation between virulence and CPS production in ccharide ,7. Similccharide . It was operties . V. vulnificus isolates also displayed resistance to the bacteriocidal action of NHS [V. vulnificus activated significantly less complement than other pathogenic Vibrio spp. [V. vulnificus are resistant to killing by NHS, while isogenic translucent variants are susceptible, which supports the hypothesis that CPS protects against serum killing [V. vulnificus cells that would otherwise facilitate the innate immune responses of the host, including phagocytosis by PMNs and macrophages and complement-mediated killing [Highly virulent n of NHS ,23. In a killing ,24. It i killing ,24. MoreV. vulnificus, such cytokines include tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), IL-6, and IL-8 [V. vulnificus cells, a potential role for it in host cytokine activation was investigated. When exposed to either an encapsulated strain of V. vulnificus or purified CPS from that strain, the production of TNF-α was stimulated in vitro in human peripheral blood mononuclear cells or in vivo in mice [V. vulnificus clinical isolate versus an isogenic CPS mutant. The encapsulated strain induced significantly greater IL-8 production and NF-κB transcriptional activity than the mutant, and TLR-2 mRNA and protein levels were also greater following exposure to the encapsulated strain. Moreover, IL-8 production and NF-κB activity were also significantly induced following exposure to purified CPS, effects which could be blocked by the addition of anti-TLR2 antibodies. Thus, V. vulnificus CPS appears to induce inflammatory cytokine production via a TLR2/NF-κB-dependent route [Septic shock typically results from excessive and systematic induction of inflammatory cytokines. For septicemia caused by and IL-8 ,3. A numand IL-8 . Given t in mice . Lee et nt route . V. vulnificus is its apparent effect on production and spatial distribution of outer membrane vesicles (OMVs) [V. vulnificus, a hypothesis supported by the finding that OMVs of this species induced death of epithelial cells by delivery of the cytolysin-hemolysin VvhA [An additional potential virulence-related function of the CPS of s (OMVs) . As withsin VvhA .Escherichia coli has served as the model for characterization of capsules from other bacterial species. Based on CPS composition, as well as the underlying arrangement and complement of biosynthetic and transport genes, capsules of E. coli have been divided into four groups [V. vulnificus have been Groups 1 and 4. Capsules within Groups 1 and 4 are also interesting in that they are related to LPS O antigens [CPS biosynthesis and genetics in r groups . To dateantigens . E. coli contain acidic sugar residues  as part of their repeat-unit structures, while Group 4 capsules are notable for the presence of acetomido sugars. For both capsular groups, individual repeat units are assembled on the lipid carrier undecaprenyl phosphate (und-P) with assembly occurring on the inner side of the inner membrane. Glycosyltransferase enzymes add the particular sugar residues that compose the repeat unit to the lipid carrier. In the case of Group 1 capsules, the WbaP protein, which is a member of the polyisoprenyl-phosphate hexose-1-phosphate transferase protein family, is the initiating glycosyltransferase, and it catalyzes the transfer of galactose-1-phosphate or glucose-1-phosphate to und-P, while WecA (polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferase family) performs the initial transfer of glucosamine-1-phosphate to und-P during Group 4 CPS synthesis. Following assembly, the completed und-P-linked repeat units for Group 1 and 4 capsules are translocated across the inner membrane by the Wzx flippase (polysaccharide transport (PST)-1 family; E.C. 7.5.2.6). The individual repeat units are then joined together on the periplasmic side of the inner membrane by the Wxy polymerase (no specified protein family), which transfers the growing polysaccharide from its lipid carrier to the newly translocated repeat unit [Group 1 capsules of eat unit . E. coli in a process that involves the Wza, Wzb, and Wzc proteins. Wza (outer membrane auxiliary (OMA) family) forms a multimeric pore through which the CPS traverses the outer membrane. Wzc is a tyrosine autokinase (and a member of the membrane periplasmic auxiliary (MPA)-1 protein family), which is anchored in the inner membrane but extends into the periplasm, and it is required for higher-level polymerization of CPS. Wzb (protein tyrosine phosphatase (PTP) family; E.C. 3.1.3.48) is the cognate phosphatase for Wzc. Both phosphorylation of Wzc and its dephosphorylation by Wzb are important for CPS synthesis [wza, wzb, and wzc genes are part of the Group 1 CPS biosynthetic and transport locus, which also includes wzx and wzy, while in Group 4 strains, the wza, wzb, and wzc genes are encoded at a separate location from the main locus [CPS undergoes further polymerization and is relocated across the outer membrane of ynthesis . Althougin locus . V. vulnificus. For strain MO6-24/O, the presence of both biosynthetic and transport genes, including wza, wzb, and wzc, at a single locus and in an apparent single long operon configuration, coupled with the acidic composition of the MO6-24/O capsule, allowed for designation of a Group 1 capsule and CPS operon for this strain [Sequencing of the genetic region flanking the sites of transposon insertions in acapsular mutants of various strains provided insights into the genetics of CPS synthesis in s strain ,32,33. wecA homolog was suggestive of a Group 4 designation, a final group determination for strain 1003(O) was not possible from the available data [Meanwhile, a partial genetic organization of the CPS locus in strain 1003(O)  was repoble data . wza, wzb, and wzc with CPS biosynthetic genes); however, the presence of a wecA glycosyltransferase gene at this locus, coupled with the CPS composition for this strain, supported a Group 4 designation [Similar analysis of strain ATCC 27562 led to the identification of its CPS locus which had characteristics of Group 1 organization .The findings of linkage between CPS transport and biosynthetic genes even for the Group 4 strain ATCC 27562 raise the possibility that such gene organization may be common in this species. As an initial test of this notion, we determined the position of the Shewanella putrefaciens strain 200 [V. vulnificus and S. putrefaciens host strains via horizontal gene transfer (HGT) events from an unrelated microbe [Interestingly, the CPS locus of strain ATCC 27562 was also found to be largely conserved and syntenous with one in the marine bacterium rain 200 . The ave microbe . V. vulnificus was explored further. It was found that V. vulnificus strains induced to a state of competence during transformation following exposure to chitin were able to incorporate into their genome exogenous DNA, including CPS locus sequences tagged with a selectable marker. In some cases, full or partial CPS locus sequences were incorporated, which resulted in a change in CPS carbotype [V. vulnificus [In a follow-up study, the potential for HGT acquisition of CPS loci by arbotype . It is plnificus  has beenlnificus .V. vulnificus strains were often readily observed [−4 to 10−5 [−4 was also observed [Both opaque and translucent colony types of observed ,9,37, th to 10−5 ,38; in sobserved . Such frV. vulnificus strains that contained a Group 1 CPS operon. They found evidence for two distinct types of translucent isolates derived from an opaque parent. One translucent type retained the ability to switch back to the opaque form, while the other type showed no evidence of switching and thus appeared to be phase locked. In electron micrographs of negatively stained cells, translucent isolates capable of phase switching showed reduced amounts of CPS on the cell surface as compared to the opaque parent, while phase-locked translucent isolates appeared to be acapsular. At the genetic level, PCR analysis revealed that phase-locked translucent isolates had typically undergone deletion of the wzb gene along with some flanking sequences within the Group 1 CPS operon . Meanwhile, translucent isolates that retained the ability for reversible phase variation (TR1) possessed no mutations at all within their Group 1 operons [Chatzidaki-Livanis et al. (2006) then studied the process of phase variation in more detail at both the phenotypic and genotypic levels for  operons . wzb. Opaque strains containing allele 2 also had multiple direct repeats present within their Group 1 transport regions, including on either side of wzb. However, the sequence of these repeats was more variable (A/CCTAGG/AAA/C) and copies were interspersed with other unrelated sequences. As shown in wzb, which would result in deletion of the intervening sequence [In the same study, examination of Group 1 operon sequences revealed a probable mechanism for the creation of phase-locked translucent variants and uncovered allelic differences at this locus. Opaque strains containing allele 1  were distinguished by having multiple copies of the same octamer sequence (ACAGGACC) repeated directly and sequentially both upstream and downstream of sequence . V. vulnificus appears to be analogous to the TR1 isolate, in that both contained an intact wzb gene and could switch back to opaque [wzb expression in newly formed Int colonies [The intermediate (Int) variant reported for certain strains of o opaque ,32,39. RV. vulnificus have now been identified. Significantly greater switching from opaque to translucent was seen during static growth as compared to growth under aeration for most strains analyzed [V. vulnificus strains, which had previously been subdivided into those types by using a randomly amplified polymorphic DNA-PCR method [A number of environmental factors affecting CPS phase variation in analyzed . Greateranalyzed . These aR method . InteresR method . V. vulnificus strains appeared translucent when grown under anaerobic conditions [wza, wzb, and wzc genes were all significantly downregulated under anaerobic conditions. These results raise the possibility that CPS production may be downregulated during earlier anaerobic stages of human infection, such as colonization of the intestinal tract [In an interesting twist on the phase variation phenomenon, it was found that opaque nditions . Howeveral tract . 2+) was found to significantly enhance colonial phase variation of V. vulnificus. Some opaque strains switched predominantly to translucent in the presence of elevated calcium while other strains switched mostly to a biofilm-proficient rugose form [2+) concentrations also significantly enhanced switching of opaque strains to translucent or rugose phenotypes [2+ or Mn2+ revealed the presence of both wzb+ translucent variants, as well as many that had undergone deletions of wzb. Thus, elevated Ca2+ or Mn2+ concentrations, which the bacterium may encounter in its natural estuarine environment, likely up-regulate existing phase variation mechanisms [Calcium  studied the ability of various V. vulnificus mutants and phase variants to colonize and persist in oysters. Reminiscent to what was seen previously in mice [V. vulnificus isolates appear to be more susceptible than opaque to phagocytosis by oyster hemocytes [In its natural marine habitat,  in mice , it was  in mice . The pre in mice , as wellemocytes .V. vulnificus. GacS/GacA is a two-component signal transduction system that regulates a variety of functions in Gram-negative bacteria, including virulence factor production [gacA knockout strain of V. vulnificus retained colony opacity, its ability to undergo phase switching to translucent was significantly reduced [gacA mutant was rpoS [S) that is critical for stationary phase gene expression. Phase variation in V. vulnificus has been purported to be a response to stationary growth [gacA and rpoS may shed light on one regulatory mechanism controlling the appearance of new phase variants in this species. Some regulatory systems also appear to affect phase variation in oduction . While a reduced . Among twas rpoS , which ey growth ; thus, trseB gene, produced mutants that appeared less opaque than the parental strain and, upon passaging, yielded mixed populations of opaque and translucent colonies at a high frequency [E, which controls expression of genes critical for maintaining cell envelope integrity during stress  [E did not recapitulate the colonial phenotype and phase-switching characteristics of rseB mutants [rseB in regulating phase variation in V. vulnificus, although the extent to which CPS expression specifically was affected in these mutants was not determined [In a separate study, insertion into or deletion of the requency . The Rseesponse) . A strai mutants . Neverthtermined .V. vulnificus. Using a fluorescence-based flow cytometry method specific for CPS, Wright et al. (1999) determined that encapsulated strains showed maximal CPS expression during logarithmic growth as opposed to the stationary phase. Moreover, opaque cells produced significantly more CPS when grown at 30 °C rather than 37 °C [While not contributing to phase variation per se, other environmental and genetics factors have also been identified that affect CPS expression in an 37 °C . V. vulnificus involves an exopolysaccharide that is distinct from CPS and is encoded by its own exopolysaccharide gene cluster [V. vulnificus [smcR mutant compared to the opaque wild type parent. A SmcR binding sequence in the promoter region for this operon was demonstrated by using DNaseI footprinting assays involving recombinant SmcR protein. Overall, the authors suggested that CPS may play an important role in regulating the size of mature biofilms by limiting their continual growth [Biofilm production in  cluster ,53. Joselnificus . Lee et l growth . In a follow-up study, CPS production and CPS Group 1 operon gene expression were found to be significantly reduced in cells subjected to heat shock at 42 °C. Basal level transcription of the CPS operon under these conditions was shown to be the result of enhanced degradation of SmcR by proteases ClpPA and Lon . ops), which is typically located upstream of the first gene within operons [ops element was previously described [V. vulnificus (rfaH gene in an opaque parental strain produced a translucent derivative that appeared phase locked. Moreover, distal, but not proximal, gene expression within the CPS Group 1 operon was significantly reduced in the rfaH mutant, and it was highly sensitive to killing by NHS. Thus, CPS production and serum survival in V. vulnificus were found to be dependent on RfaH antitermination control [Lastly, the production of a variety of surface molecules in Gram-negative species has been shown to be regulated by the antiterminator RfaH protein, which functions to prevent premature transcriptional termination at Rho-dependent sites within the cognate operons for these molecules. RfaH is recruited to the transcription elongation complex by binding a small conserved operon polarity sequence ( operons ,58. Giveescribed  within tlnificus , an effo control .V. vulnificus plays pivotal roles in circumventing the innate immune response during infection of the human host, and it appears to also stimulate a portion of the inflammatory cytokine response to this pathogen. Substantial CPS carbotype diversity among V. vulnificus strains is underscored by considerable genetic diversity, which may be the result of HGT events that lead to new combinations of CPS biosynthetic genes. As summarized in V. vulnificus should continue to provide important insights that may ultimately lead to improved disease management of this deadly pathogen.The CPS of


In the article titled “Novel Hybrid Gels Made of High and Low Molecular Weight Hyaluronic Acid Induce Proliferation and Reduce Inflammation in an Osteoarthritis In Vitro Model Based on Human Synoviocytes and Chondrocytes” , there w


A longstanding goal in science and engineering is to mimic the size, structure, and functionality present in biology with synthetic analogs. Today, synthetic globular polymers of several million molecular weight are unknown, and, yet, these structures are expected to exhibit unanticipated properties due to their size, compactness, and low inter-chain interactions. Here we report the gram-scale synthesis of dendritic polymers, mega hyperbranched polyglycerols (mega HPGs), in million daltons. The mega HPGs are highly water soluble, soft, nanometer-scale single polymer particles that exhibit low intrinsic viscosities. Further, the mega HPGs are lubricants acting as interposed single molecule ball bearings to reduce the coefficient of friction between both hard and soft natural surfaces in a size dependent manner. We attribute this result to their globular and single particle nature together with its exceptional hydration. Collectively, these results set the stage for new opportunities in the design, synthesis, and evaluation of mega polymers. Synthetic globular polymers of several million molecular weight are expected to exhibit unique properties but are difficult to synthesize. Here the authors synthesize such dendritic polymers that show unique lubrication properties and act as molecular ball bearings due to their 3D compact structure, size, solubility, hydration and low intrinsic viscosities. Linear polymers of a few million dalton (MDa) molecular weight also exhibit interesting properties, and are prepared via judicious choice of monomer and under carefully controlled reaction conditions, including reversible deactivation radical polymerization7, atom transfer radical polymerization8, reversible addition-fragmentation chain transfer9, ring-opening metathesis10, and Lewis pair polymerizations11. Synthesizing globular polymers of several million molecular weight has not been previously achieved and will, similarly, afford unexpected properties as one single polymer itself occupies a size of tens of nanometers owing to its compact structure. A path to such mega-macromolecules of several MDa molecular weight represents a significant synthetic challenge. During a polymerization reaction, the viscosity of the polymerization medium increases, which leads to the quenching of propagation species, poor control of molecular weight, polydispersity, and branching.As our mastery of polymerization reactions advances, macromolecules of increasing complexity and size are readily synthesized. Dendrimers clearly showcase this advancement; specific structural or compositional features are introduced at defined locations within a 3D globular structureHerein, we report the synthesis and characterization of semidendritic hyperbranched polyglycerols of 1, 3, and 9 MDa , and their performance as single molecule soft ball bearing lubricants on hard and soft surfaces. The mega HPGs reduce the coefficient of friction (COF) between both hard and soft surfaces, and exhibit rheological properties similar to a fluid lubricant. The high water solubility, low intrinsic viscosity, compactness, nearly molecular weight independent intrinsic viscosity, and hydration of the mega HPGs are responsible for their performance as single molecule ball bearings.13. Polymers possessing a glycerol backbone are of significant interest for biomedical and environmental-green applications due to their chemical tuneability, degradability, and biocompatibility21. We synthesized the mega HPGs, in the MDa range (up to 10 MDa), via ring-opening multibranching polymerization (ROMBP) in a single pot, using a combined macroinitiator and solvent-based solution polymerization approach  high molecular weight HPG  as the macroinitiator , mega HPG-2 , and mega HPG-3 ) by changing the glycidol to macroinitiator ratio (Table Mw for mega HPG-3 from three different batches was 9.3 ± 0.03 MDa and the yield was 74 ± 0.21%). This is significant, considering the difficulty in synthesizing semidendritic/hyperbranched polymers of such high molecular weights in large quantities, using anionic ring-opening polymerization.To overcome the challenges and prepare a large globular polymer, we used a homogenous polymerization method along with a macroinitiator to obtain ultra large dendritic polymers possessing a globular shape. As our interests lie in the polymers of potential use for environmentally responsible high-performance and biomedical applications, the selection of building blocks that are degradable, green, biocompatible, or natural metabolites is an additional key design criterionio Table . Homogen23. The degree of branching of mega HPGs, determined by 13C inverse-gated (IG) NMR spectroscopy, is between 53 and 57%, and similar to HPGs reported previously ; mega HPG-3 possesses ~389,300 bound water molecules per polymer  analysis for structure and branching density Fig. –9. A rep27, and the size does not scale with molecular weight as with linear polymers28 and low molecular weight HPG ) that range from 21 nm for the 1.3 MDa to 43 nm for the 9.3 MDa polymer Table . The aveHPG Fig. . The datη], of the mega HPGs does not change considerably with molecular weight , which show an unusual bell shape dependence of [ƞ] with increasing molecular weight  which indicates substantial back folding of terminal groups into the interior core of the dendrimer structure30. This conformational change may be occurring with mega HPGs to some degree as evident from their nearly molecular weight independent intrinsic viscosity; however, additional studies are needed to confirm this notion. Interestingly, the intrinsic viscosities of the mega HPGs are slightly increased with molecular weight compared to their lower molecular counterparts; the increase in size of the mega HPGs might be attributing to this rise31. The intrinsic viscosity values of the mega HPGs are significantly lower than linear water-soluble polymers of similar molecular weight 35, and this observation further confirms the compact nature of the mega HPGs. For example, the mega HPG-3 (9.3 MDa) possesses an intrinsic viscosity of 6.15 mL/g that is similar to most of the globular proteins in aqueous solutions38, whereas a linear PEG (Mw 11 MDa) has a value of 2600 mL/g28. Thus, mega HPGs show a unique combination of ultra-high molecular weight, compact size, and very low intrinsic viscosity almost independent of molecular weight.The mega HPGs possess very low intrinsic viscosity in comparison to other polymeric systems. The intrinsic viscosity, /[ΔHfo × MWH2O × Np][Nbw = number of water molecules bound per polymer,Hfps, ΔHfo = fusion enthalpy of polymer solution and pure water, respectively,Δw, Wtp, Wtps = weight of the pure water, pure polymer alone, and polymer solution, respectively,WtNp = number of moles of polymer taken.The morphology of the mega HPGs were visualized using cryo-SEM assembled with a focused ion beam SEM-FIB equipped with Quorum PP3010T cryo sample preparation system. A small volume of sample (10 µL) solution was loaded onto the sample carrier and immersed in slush liquid nitrogen for rapid freezing. The carrier with the frozen solution pellet is quickly vacuum transferred to the sample preparation stage cooled at −140 °C for fracturing the pellet. The fractured pellet was further transferred to the SEM stage cooled also at −140 °C for imaging the fractured surface, with an electron beam of 1 keV and 13 pA. The optimal concentration of the mega HPGs in Millipore water was 0.1 mg/mL. The average size of the mega HPGs were calculated from the ten images with ~500 particles.10. Cells were seeded at 10,000 cells/well and allowed to settle and adhere for 24 h before addition of mega HPG or control samples. Mega HPG samples (10 µL) were prepared in regular growth media (DMEM) for (90 µL) to obtain a final polymer concentration of 1.25 mg/mL. Cells were incubated with the mega HPG samples for 48 h at 37 °C. Wells containing 50% DMSO was used as a positive control. Wells containing saline and media in similar volumes to the polymer samples were used as normal controls. After incubation, cells were washed with PBS three times followed by the addition of 100 µL of fresh media and 10 µL of 12 mM MTT reagent (from ATCC). After 4 h, 100 µL of sodium dodecyl sulfate-HCl was added to solubilize the generated formazan. After solubilization for 2–24 h, absorbance at 570 nm was read on a SpectraMax 190 microplate reader (Molecular Devices) and compared to saline controls. Three technical replicates were conducted per sample, and each study was independently repeated in triplicate. Average values and standard deviation are reported.Cell viability was assessed in Tc28a2 juvenile human chondrocytes and 3T3 murine fibroblast cells using the -2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) assay as per previously reported protocolμ) was calculated from μ = (3/2) × (τ/Nr), where: τ = torque, N = load, r = plug radius62. Sampling frequency was 10 Hz.Viscosity measurements were performed on a TA Instrument AR2000 rheometer using a 2° aluminum cone and a 47 µm gap at 25 °C. Viscosity was then averaged across shear rates from 10 to 100 per s. Stribeck curves were constructed on a DHR-2 rheometer (TA instruments) with a stainless steel ring on plate tribology geometry attachment. A total of 300 µL of each lubricant were applied to the stainless steel surface and a 5 N load was applied, radial velocity increased from 0.001 rad/s to 50 rad/s while the load was held constant. This test was replicated three times for each lubricant, using fresh lubricant for all measurements. Average COF values and Hersey numbers, along with standard deviation were used to construct Stribeck curves for all formulations of mega HPG along with Synvisc One and BSF as controls. The values of 7 and 23% for mega HPGs were chosen as to ensure a 3× difference in amounts to better observe potential differences. Cartilage on cartilage COF measurements, using 7 mm diameter bovine osteochondral plugs, were performed on a Bose Electroforce 3200. Cartilage plugs were cored from skeletally mature bovine knees using a diamond tipped drill bit. Plugs were incubated in 0.5 mL of lubricant at RT overnight prior to testing. An 8 N (200 kPa) creep load was applied to paired osteochondral plugs. After 3 h of compression to equilibrate the tissue in creep, equilibrium friction measurement was made. Friction measurements used an angular velocity of 360°/s (effective velocity = 14.7 mm/s) for 120 s. COF . The contact mode was applied using a silicon nitride tip with a nominal spring constant of 40 pN/nm. Mega HPG-3 was chemically adhered to the surface of epoxide functionalized glass slides. A total of 5 mg of mega HPG-3 was diluted in 50 mL of dimethylformamide. Stoichiometric amount of sodium hydride was then added. Ten microliters of this solution at concentration of (0.1 mg/mL) was dropped on the surface of the epoxide glass slide and kept at 60 °C overnight. The glass surface was rehydrated in PBS just before experimentation. The tip was slowly lowered to the surface to confirm contact without damage. Tip calibration was done on the bare glass surface, which acts as an infinitely hard surface, to set both a baseline deflection and virtual deflection correction. This determines the cantilever’s inverse optical lever sensitivity . The spring constant was verified via thermal tuning and was always within the range dictated by Bruker (within 30 pN/nm). After calibration, an area scan was performed in tapping mode prior to indentation to confirm the presence and density of the polymer. Clumps of polymer were chosen for indentation based on shape and size. Force spectroscopy was then obtained over a 2 μm extension length and a 1000 nm/s approaching and retreating velocity. The tip was triggered at a set-point of 0.75 V of deflection. Once an indentation was performed, the raw distance of the tip along the Supplementary InformationPeer Review File


We show how known AMR protein variants tend to correspond to exposed residues, while on the contrary their susceptible counterparts tend to be buried. Based on these findings, we develop RSA-AMR, a novel relative solvent accessibility-based AMR scoring system. This scoring system can be applied to any protein variant to estimate its propensity of altering the relative solvent accessibility, and potentially conferring (or hindering) AMR. We show how RSA-AMR score can be integrated with existing AMR detection algorithms to expand their range of applicability into detecting potential novel AMR variants, and provide a ten-fold increase in Specificity. The two main limitations of RSA-AMR score is that it is designed on single point changes, and a limited number of variants was available for model learning.Antimicrobial resistance (AMR) is a significant and growing public health threat. Sequencing of bacterial isolates is becoming more common, and therefore automatic identification of resistant bacterial strains is of pivotal importance for efficient, wide-spread AMR detection. To support this approach, several AMR databases and gene identification algorithms have been recently developed. A key problem in AMR detection, however, is the need for computational approaches detecting potential novel AMR genes or variants, which are not included in the reference databases. Toward this direction, here we study the relation between AMR and relative solvent accessibility (RSA) of protein variants from an  Mycoplasma genitalium from urethral swabs or urine sediments  is a significant and growing public health threat. Treating infections caused by resistant organisms is clinically challenging, and sometimes impossible; even when resistant infections can be treated with alternative antibiotics, these treatments are often costly both in terms of healthcare costs as well as increased morbidity in treated patients. For these same reasons, AMR is a significant challenge in veterinary and plant health, and the need to develop novel AMR treatments is deemed very urgent . Accordiediments ; Neisseral swabs ; Klebsied sputum ; and Camal cases .Systems for automated AMR identification such as AMRPlusPlus , Meta-MAB, b, e, and E. The classes are described as follows: B, very buried (less than 4% exposed); class b, somewhat buried (between 4 and 25% exposed); class e, somewhat exposed (between 25 and 50% exposed); and class E, very exposed (more than 50% exposed). For each residue, the scores predicted for all the classes represent a probability distribution, therefore their probability sum is bound to be 1.Briefly, Brewery  is a staBacillus subtilis pgsA with mutation conferring resistance to daptomycin is annotated as A64V, meaning that a Valine in position 64 confers the described resistance. In addition to the variant annotation, CARD reports the susceptible protein sequence as well. For our analysis, if the susceptible residue in the annotated position differed from the one reported in the sequence, we considered both as susceptible. This procedure led to the collection of 1,234 resistant protein variants out of 152 susceptible proteins.We extracted the protein variants from the CARD protein variant model, considering single resistance variants only. To describe the variant, CARD reports position, susceptible (wild type) amino acid, and resistant one. For example, a record of a resistance variant for These data are used firstly to measure the difference in the RSA classes between resistant and susceptible variants, and then to elaborate the RSA-AMR score.resistant to a specific AMR molecule or mechanism, and it is well and extensively aligned against a PATRIC protein from a proteome which is susceptible to the same AMR molecule or mechanism, then the alignment mismatches can be read as candidate variants involved in the resistance loss. Conversely, candidate variants  found in proteins from PATRIC resistant proteomes are more likely to not affect the resistance, as both the MEGARes and PATRIC proteins are annotated with the same AMR resistance.CARD AMR protein variants typically belong to housekeeping genes. To extend our study to a more general case, we extracted candidate AMR and susceptible protein variants by aligning AMR MEGARes proteins with both AMR and susceptible proteomes downloaded from PATRIC . We seleMEGARes V2.00 genomic sequences were obtained and AMR variant-dependant accessionsed were filtered out (7375 sequences retained). We translated MEGARes sequences into peptides with ORFfinder , consideWe then searched for specific terms to extract drug-specific resistant MEGARes sequences, and drug-specific resistant and susceptible PATRIC proteomes, namely tetracyclines, ciprofloxacin, ampicillin, and amikacin. Our rationale for choosing the PATRIC labels used in this work was based on three criteria, namely (a) abundance of the labels in both databases; (b) one-to-one non-ambiguous mapping between MEGARes hierarchical nomenclature and PATRIC labels; and (c) labels that confer resistance to antimicrobials listed as critically important for human medicine by P-values comparing the score distributions have been calculated as pairwise (resistant versus susceptible) Wilcoxon signed-rank tests. P-values for probability distributions from candidate protein variants derived from MEGARes and PATRIC are calculated as Wilcoxon rank sum tests.For CARD protein variant RSA probability distributions, To show how the RSA-AMR score can ameliorate the performance of existing AMR detection algorithms, we used it in combination with Meta-MARC, DeepARG, and AMRPlusPlus. We obtained a set of resistant-susceptible gene pairs as follows. We considered all the candidate susceptible protein variants derived by aligning MEGARes versus PATRIC, as in Sections 2.3 and 2.4. For each susceptible protein variant, we calculated all the possible corresponding nucleotide codons, and obtained a candidate susceptible gene for each codon. We coupled candidate susceptible protein variants with their reference resistant gene on MEGARes obtaining a balanced data set of resistant and candidate susceptible instances. Regarding classification for Meta-MARC, we considered the lowest E-score for each sequence; for DeepARG, we considered the DeepARG AMR probability output for each sequence; for AMRPlusPlus we considered the resistome, i.e., the sum of the AMRPlusPlus BWA alignements to the reference database.B (very buried), b (somewhat buried), e (somewhat exposed), and E (very exposed). We found resistant variants show a higher probability than susceptible ones in classes e (medians 0.23 versus 0.2), and E (medians 0.24 versus 0.06). Conversely, susceptible protein variants show a higher probability in class B when compared to resistant ones . We did not find a variation in the medians for class b (both 0.35).We calculated the solvent accessibility for each protein, susceptible and resistant, from 1,234 resistant protein variants obtained from the CARD . The BreClostridioides difficile resistance to fluoroquinolone, and six Mycobacterium tuberculosis resistances . We found the RSA behavior to be consistent also if we split the variants by mechanism. Class B is invariantly scoring higher for susceptible protein variants, while classes e and E for resistant ones. Differently from the general case, class b is scoring higher for susceptible protein variants in four out of six molecular mechanisms.We investigated the solvent accessibility difference between susceptible and resistant variants across specific AMR mechanisms . We retaBy considering resistant and susceptible CARD AMR variants as classes, we developed a model based on logistic regression, with the four RSA values as features. We used the probability of this model as a RSA-based AMR protein variant score (RSA-AMR score). We measured the area under the ROC curve (AUC) for a 10-fold cross validation for logistic regression (AUC 0.55), J48 decision tree (AUC 0.55), and CART (AUC 0.67) decision tree, as depicted in We extracted candidate AMR and susceptible protein variants by aligning AMR MEGARes proteins with both AMR and susceptible PATRIC proteomes.variations of the RSA probability distributions (Δs), as the difference between the resistant (MEGARes) solvent accessibility distribution; and the candidate resistant or susceptible variant (PATRIC) solvent accessibility distribution. ΔB, Δb, Δe, and ΔE are calculated as MEGARes residue score (resistant) – PATRIC score. Therefore when we consider genes from susceptible PATRIC genomes, we expect a change in the score; whereas when we consider genes from resistant PATRIC genomes, we expect a negligible score change, ideally ΔB, Δb, Δe, and ΔE = 0.When examining CARD variants, we use two sets of RSA probability distributions, i.e., the ones for the susceptible (wild type) variants, and the ones for the resistant variants. For candidate variants extracted from the MEGARes versus PATRIC alignment, on the other hand, we have to consider three sets of RSA probability distributions. Specifically, we calculate the RSA for the resistant  residues from MEGARes; the RSA for the candidate resistant variants; and the RSA for the candidate susceptibility variants. We therefore do not compare RSA probability distributions but E, where the median Δ is negative. For susceptible protein variants, three out of four classes  show Δ≠ 0, suggesting a structural variation. In ampicillin  can be implicitly related to their ability to retain (or lose) AMR against specific substances.In tetracyclines , resistapicillin , the medpicillin , while tTo show how it is possible to utilize the RSA-AMR score to expand the range of applicability of existing AMR detection algorithms, we show three case studies with state-of-the-art algorithms, namely Meta-MARC, DeepARG, and AMRPlusPlus. We derived a set of resistant and susceptible genes by pairing candidate susceptible variants described in Section 2.6. We chose this set as we can not use CARD since the RSA-AMR score is based on those very same variants. Resistant genes from Megares are paired with candidate susceptible ones, i.e., carrying a variant obtained from PATRIC alignments. Current algorithms are not designed to answer the specific task represented by our data set, which would be to detect susceptible sequences from their resistant counterparts based on a single protein variant per sequence, as their AMR labeling is based on sequence similarity (for example percent of identity with sequences in the reference database). We observed that these algorithms classify the great majority of the candidate susceptible variants as resistant . We show how using the SRA-AMR score can greatly ameliorate the results and greatly curb the number of false positives, increasing specificity.Integration with Meta-MARC. The Meta-MARC predicted resistome depends on a user-defined E-value threshold. To explore the integration of the RSA-AMR score with Meta-MARC, we tested how Sensitivity and Specificity vary according to the E-value threshold, and how the RSA-AMR integration can ameliorate the overall performance , but at the cost of a sharp decline in Sensitivity . Thus, Meta-MARC cannot accurately distinguish genes carrying the candidate susceptible variant from the resistant ones, as it tends to classify both classes as resistant. We used the RSA-AMR score to balance Sensitivity and Specificity in an ensemble (stacking) fashion. We considered the Meta-MARC predictions for the resistant class as true; however, for the susceptible class, we considered the Meta-MARC predictions true only if the associated RSA-AMR score is positive, i.e., predicted positive by the RSA-AMR logistic regression. When compared to the Meta-MARC prediction for the most stringent threshold of 10–25, Specificity is greatly increased (0.05–0.49), with a minor loss of Sensitivity (0.63–0.6).formance . We filtIntegration with DeepARG. The same data set utilized for the Meta-MARC integration was also tested with DeepARG. We varied the DeepARG classification threshold from 0.8 (default) to 0.95  decreases by 65% , while the alignements on the resistant class (true positives) decreases only by 38% .RSA-AMR as a meta-classfier. We assessed the potential of integrating Meta-MARC, DeepARG, and the RSA-AMR score in a meta-classifier. The meta-classifier is an AMR variant resistance classifier that accepts the outputs of single classifiers as input. We considered the filtered resistant/candidate susceptible data set to train meta-classifiers based on logistic regression, J48, and CART. As features for the meta-classifiers we considered the RSA-AMR score, the Meta-MARC E-value, and the DeepARG classification probability, or a combination of them. The main state-of-the-art algorithms used to predict AMR in unknown sequences are based on alignment with known AMR genes , and areb and B, and conversely positive Δ for classes e and E. However, this does not hold for other AMR mechanisms, where we notice a predicted structural variation in the candidate susceptible variants in both directions. There are several factors that, alone or combined, can explain why the shifts are in both directions. One possible explanation is that we use single-point mutations, whilst more extensive combinations of mutations could be needed for significant structural changes. However, when analyzing the so-called housekeeping genes that are known to confer AMR with exactly one mutation, we clearly observe a definite, strong shift in Δs between the E and B categories. Another possibility is that different antibiotics have different binding affinity and mechanisms of action, such that some might be less affected by structural changes, particularly if their mechanism of action doesn’t involve binding in that portion of the protein. Furthermore, the resistance mechanism for some proteins might in fact be due to burying of the binding site itself, i.e., by exposing a specific residue the resistance is hindered because the target antibiotic can bind. In the case of active translation, the protein blocks the binding site without preference for the direction of the change, depending on drug and mechanism. Finally, we are conducting in silico experiments based on predicted (not observed) data, i.e., predicted secondary structure and candidate protein variants. Therefore our data are strong enough to contain the biological signal connecting changes in solvent accessibility to changes in AMR, but are not sufficient to define its direction.Consistently with these findings, we did not find a strong shift when measuring the ΔRSA in candidate resistant protein variants obtained by comparing resistant PATRIC proteomes and MEGARes resistant genes . On the other hand, the shift was present in the candidate susceptible protein variants, indicating a structural change. Intuitively, this change can be interpreted with the fact that a structural change is altering the AMR properties, specifically diminishing or removing AMR. However, the shift greatly varies according to the specific AMR mechanism  in termsad hoc manual curation. While MEGARes provides a lookup table matching its sequences with the CARD ones, the AMR mechanism description is fundamentally different in the two databases. MEGARes structures each sequence into type, class, mechanism, and group; CARD provides an ontology-oriented labeling. PATRIC is instead annotated with AMR molecule-specific names. PATRIC annotations are not always consistent, with the same AMR molecule labeled with multiple names, or genomes showing records as both resistant and susceptible to the same AMR molecule (see Methods). Although designed for single variants, the RSA-AMR score can be applied in principle to proteins showing multiple variants, e.g., in an additive fashion. However, multiple non-synonymous changes can significantly impact the protein structure. In turn, structures computed by prediction tools might not be reliable when calculated upon multiple changes, possibly introducing larger errors. We plan to address a multiple-variant model in our future works.A challenge in our experiment design was to match CARD, MEGARes, and PATRIC annotations, which required In conclusion, in this study we analyzed AMR protein variants from the RSA perspective. We analyzed the RSA of AMR protein variants with Brewery, a state-of-the-art method based on deep learning. We found a strong distribution shift in resistant residues, with respect to RSA, when compared to the susceptible ones. Our approach unveiled RSA differences also in putative resistant and susceptible variants, obtained by aligning MEGARes and PATRIC sequences. These differences are measured as the variation in the RSA probability distribution. We found these differences varying greatly depending on the considered AMR machinery. Based on these findings, we developed a novel scoring system, RSA-AMR. RSA-AMR is not meant to be used as a standalone AMR prediction tool, but rather as a variant screening. When combined with state-of-the-art classifiers, it provides a ten-fold increase in predictive Specificity . Limitations of RSA-AMR include the fact that it works on single point changes, and a limited number of identified protein variants was available for model learning.https://github.com/smarini/RSA-AMR.The datasets generated and analyzed for this study can be found in the RSA-AMR github page. MP, CB, SM, IBS, and NRN conceived the project. SM and MP designed the experiments. SM and MO conducted the experiments. SM, MP, and CB wrote the manuscript. All authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A critical issue receiving much less attention during this pandemic is the effect it could have on antimicrobial resistance (AMR). With no proven therapy for COVID-19, prescribers are more likely to use antibiotics indiscriminately for treatment and prevention of presumed bacterial co-infections. In high-income settings, where bacterial cultures can be done on a timely basis, such antibiotic regimens can be stopped within 48 hours. However, in Africa, unneeded empiric antibiotic regimens are likely to be continued for a longer period of time due to lack of bacterial culture and antimicrobial susceptibility testing (AST) capabilities. In addition, the widespread use of hand sanitizers and antimicrobial soaps could exacerbate AMR among healthcare workers and the general population. In this article, we discuss how the COVID-19 pandemic could affect the already precarious AMR situation in Africa and discuss ways to minimise the effects on public health.The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in 7 145 539 confirmed cases and 408 025 deaths by 03 June 2020.3 in the era of COVID-19. It has been estimated that AMR could lead to 10 million deaths by 2050 if nothing is done about the menace, and most of the deaths are likely to occur in Asia and Africa.4 Actions such as infection prevention and control, antibiotic stewardships, capacity building (laboratory infrastructure and personnel) and surveillance are necessary to ameliorate the impact of AMR. To support a global action plan on AMR, the World Health Organization has developed the Global Antimicrobial Resistance Surveillance System to provide a standardised approach for collection, analysis and sharing of data related to AMR to inform decision-making at national and international levels; surveillance of AMR bacteria in humans and animals across the globe is key in tackling the problem of AMR.5 Unfortunately, this is hampered by the limited data from Africa due to limited diagnostic microbiology infrastructure on the continent.6Antimicrobial resistance occurs when microbes change and render antimicrobial agents ineffective. This phenomenon is fuelled by overuse and misuse of antimicrobial agents in humans and animals. Antimicrobial resistance leads to treatment failures, prolongs hospital stays, worsens clinical outcomes and makes surgical procedures and chemotherapy risky and unsafe. With increasing antibiotic resistance and few new antimicrobial agents in the production pipeline, it is imperative to monitor the epidemiology of bacteria species, especially respiratory pathogens to inform treatment decisionsStreptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus, resulting in increased severity and mortality.7 The majority of deaths from the 1918 influenza pandemic resulted from secondary bacterial infections.8 During the 2009 swine influenza pandemic, there was an increase in hospital pneumonia cases as a result of secondary bacterial pneumonia, which resulted in 29% – 55% of deaths.9 In a recent study on the outcome of COVID-19 patients from the Wuhan region of China, half of the non-survivors had secondary infections.10 Of note, the majority (95%) of the patients in the study were given antibiotics.10Viral respiratory infections can be complicated with secondary bacterial infections, commonly with 12 without much evidence to support its use. It is worth noting that the World Health Organization has warned against indiscriminate use of antibiotics during COVID-19 treatment.2Azithromycin, a broad-spectrum macrolide antibiotic (in combination with hydroxychloroquine) has become a common treatment for COVID-19 patients in several parts of the world including AfricaS. aureus infections in hospitals, which was linked to heavy use of antimicrobial agents.14 It is therefore possible that COVID-19 patients could also be battling with AMR bacteria, in addition to the virus, resulting in poor patient outcomes. Urgent studies are needed to define the bacteria aetiologies and AMR bacteria that contribute most to mortality in COVID-19 patients.While antibiotics may not affect COVID-19 directly, they are agents administered to prevent or treat secondary bacterial infections in COVID-19 patients. Therefore, the swell in the numbers of COVID-19 patients could lead to the inappropriate use of antibiotics and subsequent selection of AMR bacteria. In previous epidemics, there were indications of an increase in methicillin resistant 15 More research in Africa is needed to determine how the widespread use of antimicrobial sanitizers and soaps affects the microbiome and contributes to AMR.From the onset of the pandemic, the general public has been constantly directed to regularly wash hands with soap and water, and use hand sanitizers. Although such practices will help improve hygiene standards and reduce the spread of COVID-19, they could have negative effects on AMR. Disinfectants and antimicrobial soaps contain biocides or antimicrobials. An increase in use of these agents may lead to an increase in their concentration in waste water and receiving water bodies; this may have a potentially negative impact, because the elevated concentrations may lead to selection of AMR bacteria, posing a health risk to persons exposed to such environments.16 A survey indicated that very few countries in Africa have functional national surveillance systems for AMR of common bacterial infections from community and hospitalised patients.17 An external quality assessment report indicated poor performance of AST in several African countries.18 Quality assurance in AST is key for reporting and implementation of the Global Antimicrobial Resistance Surveillance System19; laboratories in Africa have to be supported with the needed investments for better performance.The World Health Organization reports that there is limited data on AMR prevalence from Africa due to limited laboratory capacity and surveillance networks.16 In the absence of microbiology and AMR data, prescribers often manage clinical symptoms rather than specific bacteria,16 a situation that may fuel inappropriate use of antimicrobials and emergence of resistant microbes.With the paucity of data on AMR, realistic evidence on how AMR may compromise first-line empirical treatment in common bacterial infections is lacking on the continent.20 For the few laboratories that are able to do culture and AST, performing such tests in a standard way is a challenge often due to a lack of reference strains and up-to-date standard interpretation guidelines. In addition, culture of bacteria and AST are often not requested by clinicians, because of cost to patients and the long turn-around time that often render the results useless for patient care. Altogether, data on AMR bacteria to guide treatment decisions at local and national levels are scarce. Newman et al.20 conducted the first nationwide AMR surveillance in Ghana between 2002 and 2003 and found bacteria species resistant to ceftriaxone (6.3%) and ciprofloxacin (11%).21 Opintan et al. (2015), followed up with another nationwide survey between June 2014 and November 2014 and observed that > 50% of the commonly isolated bacteria species were resistant to third-generation cephalosporins and fluoroquinolones. In this study, the majority of the Gram-negative bacteria species recovered were positive for extended spectrum beta lactamase; these organisms are resistant to a wide range of antimicrobial agents.22In Ghana, like most African countries, the majority of microbiology laboratories do not have the capacity to perform culture and AST tests.Escherichia coli strain resistant to meropenem and belonging to ST410 was detected in a urine sample from a hospitalised patient in the northern part of Ghana. In this first report, the detected plasmid co-carried other resistance genes.23 This is disturbing, mainly because these organisms are resistant to the antimicrobials used as last-line treatment for severe bacterial infections.24In another study, a New Delhi metallo-beta-lactamase-producing µg/ml) belonged to ST1407, a pandemic resistant clone.25Among female patients who presented with vaginal discharge, dysuria, intermenstrual bleeding or abdominal pain and men who presented with urethral discharge or dysuria, gonococcal isolates recovered from samples collected from five healthcare centres were resistant to tetracycline (100%), benzylpenicillin (91%) and ciprofloxacin (82%). One isolate resistant to cefixime  and 916 clinical isolates  in other studies; methicillin resistant S. aureus isolates detected belonged to global epidemic clones, including USA300.29A total of 520 methicillin susceptible 16 The majority of AMR data in Ghana are from pockets of studies focusing on particular bacteria species recovered in select hospitals or research institutions. They therefore may not reflect the national AMR situation and could be an underestimation of the actual magnitude of the AMR problem in a country where self-medication is rampant and antimicrobial agents are often available without prescription.30Our review of more than 20 articles on AMR from Ghana for this write-up revealed that resistance of bacteria species  to commonly used antimicrobial agents such as ampicillin, tetracycline, chloramphenicol and trimethoprim sulfamethoxazole was common. The situation is not different from other parts of the continent.5 Ghana also has a national AMR working group; the group meets regularly to deliberate on AMR issues in the country. To strengthen knowledge and evidence base through surveillance and research, Ghana received the first Fleming Country Grant, which is currently supporting a government-led system of collecting, analysing and reporting of AMR and antimicrobial use data from humans and animals from 11 sentinel sites. These data will provide a national AMR picture to inform treatment decisions at the national level. Plans are underway to begin a pilot surveillance study.Ghana is gradually gathering momentum to get to a stage of having a functional national system to monitor AMR. The Antimicrobial Use and Resistance Policy and the national action plan on AMR, which provide strategies and plans to guide AMR data generation for evidence-based interventions at local, national and international levels, were launched by the President of Ghana on 11 April 2018. The national action plan was fashioned along the lines of the objectives of the Global Action Plan on AMR.The prevalence and mortality rate of COVID-19 in Africa is lower compared to that of Europe, America and Asia. However, the rapid spread of the virus is another call to strengthen the laboratory diagnostic capacity in Africa to perform standard antimicrobial susceptibility testing, especially of relevant respiratory pathogens to inform treatment decisions in healthcare facilities. Importantly, AST data need to be collected yearly to support empiric treatment in hospitals. Continuous surveillance is required on the African continent and across the globe to understand the epidemiology of AMR pathogens, provide the needed data to guide and inform treatment decisions and policies and monitor resistance trends and emergence of new clones. In all of these actions, the role of networks and collaborations in enhancing the success of various interventions cannot be overemphasised. Ownership and sustainability plans by governments and local authorities are key to maintaining the successes for continuous AMR surveillance activities with local and international partners. Capacity building (infrastructure and personnel) is a must-have and must be continuously improved to fight AMR in Ghana and Africa.S. aureus. In addition to phenotypic methods used in the detection of resistance, genomic tools such as whole genome sequencing can be utilised to generate extensive data to expand our knowledge on the changing epidemiology of AMR bacteria. During the COVID-19 pandemic, urgent studies are needed to document the bacterial organisms responsible for co-infections at the local level to guide empiric therapy. Due to the widespread use of hand sanitizers and antimicrobial soaps, research is needed to study healthcare workers and others for changes in the skin flora that they carry, which could be transmitted to vulnerable patients. Healthcare institutions without antibiotic stewardship programmes must take steps to institute measures for rational antibiotic use. Antibiotic stewardship optimises institutional antibiotic use and reduces the selection and spread of AMR; similarly, infection prevention needs strengthening across the board to limit the spread of resistant bacteria. Finally, the need for increased investment by African governments to drive development of new diagnostic tools, novel antimicrobial agents and vaccines cannot be over-emphasised.One cannot blame the African prescribers for throwing the ‘kitchen sink’ of antibiotics at sick and dying COVID-19 or other virally infected patients. Rather, these practices could be reduced significantly, if physicians were empowered to order cultures and obtain results in a timely manner. Doctors faced with negative culture results are more likely to stop antibiotics than if no results are available. With the necessary political will, African governments and academics can do a few things in line with the five strategic objectives of the Global Action Plan on combating AMR during the current pandemic and beyond. Firstly, there is a need to raise awareness of AMR among personnel in human and animal health, and agriculture, as well as among consumers, to ensure a proper understanding of AMR pathogens and the impact of AMR across sectors. There is an urgent need to build laboratory capacity to generate the required microbiology data through surveillance and research to inform treatment decisions, especially in urban centres where resistant organisms are often abundant. Evidence-based prescribing and dispensing should be the way to go to optimise antimicrobial use. More automated systems like the GeneXpert platform used for tuberculosis could be repurposed for organisms like methicillin-resistant The AMR menace has been described as a problem that knows no borders; resistant bacteria can be found in humans, animals, food and the environment. The current pandemic shows that we remain susceptible to infections for which we have no therapeutic options. The experience from COVID-19 therefore should be another reminder of the life-threatening consequences of AMR microbes, and the need for rapid capacity building (infrastructure and human resources) for surveillance of resistant bugs on the African continent.


One of the key strategic objectives of the World Health Organisation’s global antimicrobial resistance (AMR) action plan is to improve public awareness and understanding of this issue. Very few AMR awareness campaigns have targeted the animal production sector, particularly in low- and middle-income countries (LMICs) where rural communities can be geographically difficult to access via traditional face-to-face community engagement methods. Aquaculture is a major food production industry in Bangladesh and across Asia, an area which poses a significant risk to global AMR dissemination. In this pilot study, we sought to investigate the potential for digital communication materials to rapidly and effectively communicate AMR messages to rural aquaculture farmers in Bangladesh. Working with stakeholders from the Bangladesh aquaculture industry, we developed a 4-minute digital animation designed specifically for this audience and assessed its capacity to engage and communicate AMR messages to farmers. We then conducted a small-scale social media campaign, to determine the potential for rapidly disseminating AMR awareness materials to a large audience across Bangladesh, where there is an extensive 4 G internet network and an ever-increasing proportion of the population (57% as of December 2019) have mobile internet access. Thirty-six farmers were surveyed: all of them liked this method of communication and 97% said it would change the way they use antibiotics in the future. Through the social media campaign, the animation received 9,100 views in the first 2 weeks alone. Although preliminary, these results demonstrate the huge potential for digital communication methods for the rapid and widespread communication of AMR awareness materials to rural aquaculture communities in Bangladesh and across Asia. Our results support the need for more research into the most appropriate and effective content of AMR awareness campaigns for aquaculture communities and question the need for explaining the science underlying AMR in such communication materials. The overuse of antibiotics in animals in low- and middle-income countries (LMICs) is a serious issue that is contributing to the increasing global burden of antimicrobial resistance (AMR) ,2. AlthoSouth East Asia has been identified as the global area posing the greatest risk to AMR dissemination . In thishttps://www.youtube.com/watch?v=P_mnoK5E53Q (Bangla) and https://www.youtube.com/watch?v=zR0EB9P7rKc (English).In order to establish the most appropriate messages to convey to a Bangladesh aquaculture audience, we consulted with a range of aquaculture industry stakeholders and community engagement practitioners at an ‘AMR, One Health and Aquaculture’ workshop, held in Dhaka, Bangladesh, in February 2019 ,13. The 2; 92%), and farmed a combination of shrimp, prawn and fish species (91%) (Supplementary Information). Farmers were then shown the animation on a tablet, and feedback was collected through a questionnaire. All data were collected using the SurveyMonkey app.In May 2019 we visited 36 aquaculture farms  in two of the major aquaculture-producing districts of Bangladesh, Khulna (20 farmers) and Bagherat (16 farmers), to seek feedback on the animation. Before showing farmers the animation, we collected preliminary information on their behaviour, in terms of access/use of medicines and levels of awareness of AMR. The majority of people surveyed (83%) were male, and most of the farms were extensive (97%), small-medium sized  with information on the animation and links to the English/Bangla version of the animation was also distributed to 364 people at 185 organisations, including media outlets, Bangladesh Government, aquaculture-related businesses, non-governmental organisations, donor organisations and educational institutions. An English version of the animation was posted on the WorldFish and CGIAR  global twitter accounts with 97,900 impressions (the number of twitter feeds it was displayed in) and 11,200 views.On 30 May 2019, the animation was released via the WorldFish Bangladesh Facebook channels. Within Bangladesh, Facebook is the most widely used social media platform, and between this date and 5 December 2019, the Facebook page containing the Bangla version of the animation was viewed 17,614 times. On 1 June 2019, a targeted email marketing campaign press release  of farmers said they understood the animation clearly ). When aPrior to being shown the animation, none of farmers claimed to have heard of ‘antibiotic resistance’ or ‘drug resistance’, and after viewing 97% said that it would change the way they use antibiotics in the future . We did not question whether this was an increase or decrease in usage, since it is difficult to obtain reliable information on shrimp farmers’ use of antibiotics due to the growing pressure within the industry to reduce antibiotic usage in line with export antibiotic residue checks ,14.Dissemination of the animation via a social media campaign resulted in 17,614 Facebook views (Bangla version) and 11,200 Twitter views (English version) in a 6-month period. Analysis of the Facebook metrics showed that the vast majority of the viewers were male (94%), under 35 (76%), and accessed the animation via a mobile app from the Dhaka division of Bangladesh , reflectThe international AMR community now acknowledges that, for maximal effect, large-scale awareness campaigns should be integrated within industry-wide AMR behavioural policy strategies ,17. Our This study supports others that have shown an urgent need for more research into the most appropriate content of AMR awareness campaigns, to ensure that they achieve their intention to effect behavioural changes ,19. FutuAn important limitation of our study was that Facebook does not disclose information on viewer’s profiles; therefore, we could not identify whether the 17,614 views were by people within the Bangladesh aquaculture industry. This could be addressed in future studies by linking a survey to the animation or by creating a closed Facebook group made up of the WorldFish followers based in Bangladesh, which would allow us to extract user information and be more specific in our campaign targeting. If we are successful in validating social media as a viable route for communication with aquaculture communities, then the potential impact of digital communications is considerable: interactive communication materials could be used for two-way dialogue with audiences that are very difficult to otherwise reach, allowing them to engage with industry and academic stakeholders. Accessible online courses tailored to these rural communities could also offer unprecedented access to resources that have previously been restricted due to financial, social, cultural or geographic constraints, and these could easily be translated into different languages, allowing international organisations to leverage materials and better disseminate them across regions that face similar problems. This is particularly relevant for the global aquaculture industry, with 89% of production occurring in countries neighbouring Bangladesh who face similar borderless challenges from climate change, disease and environmental pollution .This pilot study demonstrates the potential for digital media as an effective tool for the rapid and widespread communication of AMR messages to rural Bangladesh aquaculture farmers and could be used to target other rural LMIC communities in Bangladesh and beyond, who are similarly challenging to reach. Importantly, our data also support the urgent need for more research into the most appropriate content for digital AMR awareness campaigns, since with the enormous audiences involved it is imperative to ensure that they are as effective as possible in achieving their intended outcome.Click here for additional data file.


This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid , respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor  Vascular calcification is a highly regulated process observed during aging, hypertension, diabetes, and particularly chronic kidney disease (CKD). It contributes to elevated risk of cardiovascular events and mortality ,2,3,4. DEPHX2, which induces a decrease in sEH activity in vitro, suggesting a deleterious impact of EETs on vascular calcification [Increasing experimental evidence shows that the endothelium not only is a source of osteoprogenitor cells but also regulates vascular calcification through the release of signaling molecules such as nitric oxide (NO), which prevents VSMC osteochondrogenic differentiation ,6,7. Endfication ,11,12. Ification .The aim of the present study was to assess the role of EETs and sEH in the vascular calcification process using mineralization assays of rat aortas and human VSMCs, and complementary experiments on human calcified carotid arterial materials were performed.To evaluate the impact of sEH hydrolase and phosphatase inhibitors ex vivo on vascular calcification, we cultured isolated rat aortas in normal and high-phosphate conditions as previously described ,15,16. Wtrans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB), a potent and specific inhibitor of sEH hydrolase [t-AUCB was confirmed by the decrease in the 14,15-DHET-to-14,15-EET ratio  [t-AUCB   prevente [t-AUCB A, and thg effect B,C withog effect . Fluconag effect D and in   and conducted in accordance with the Directive 2010/63/EU of the European Parliament. For ex vivo mineralization assays, 10-weeks-old male Wistar rats  were euthanized by intraperitoneal injection of pentobarbital (120 mg/kg), and the thoracic and abdominal aortas were dissected as previously described ,15. AfteHuman arterial tissues were obtained from patients undergoing aortic surgery or carotid endarterectomy who gave written informed consent, and studies were conducted in accordance with the Principles of Good Clinical Practice and the Declaration of Helsinki and were approved by the local ethical committee .2 for 14 consecutive days. The medium was changed every 2 to 3 days.Aortic VSMCs were isolated from aortic lesion-free explants, as previously described and validated ,23,24. FCarotid endarterectomy samples from each patient were collected in the surgery room and immediately dissected in two fragments: the atherosclerotic calcified plaque and the distant macroscopically intact tissue. Each fragment was immediately frozen in liquid nitrogen and, intrapatient comparison of gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST arrays  as previously described .p < 0.05 was considered statistically significant.Statistics were performed using the SYSTAT package . Results are presented as mean ± SEM. For ex vivo rat aortic and in vitro human VSMC mineralization assays, all experiments have been performed using at least 3 different aortic tissues from rats or humans. Analyses of the differences between two groups were performed by student paired t test or Wilcoxon matched paired test for nonnormality distributed data. Differences between 3 or more groups were analyzed using a generalized linear model with group as the factor and the rat or human aorta used as the cofactor, followed by Tukey–Kramer multiple comparison tests for post hoc analysis. Pearson correlation analyses were used to assess the relationships between human carotid mRNA expression levels of genes of interest. A value of


Providencia spp. from Raw Cow's Milk in Baghdad, Iraq” [In the article titled “Isolation and Molecular Identification and Antimicrobial Susceptibility of d, Iraq” , the namAdditionally, there was an error in the second sentence of the abstract and the second sentence of the Materials and Methods section, where the word “McConkey” should be corrected to “MacConkey.”