Chronic wound treatment currently relies heavily on visual assessment by clinicians; however, the clinical signs and symptoms of infection and inflammation are unreliable in chronic wounds. The specialty of wound care has witnessed the advent of advanced interventions, such as cellular and/or tissue based products (CTP). The success of advanced therapies relies on preparing the wound bed by reducing bacterial burden and inflammation. The lack of diagnostics in chronic wound care leads to uncertainty in the adequacy of wound bed preparation. Recent research suggests that two novel point-of-care diagnostic tests can assist in the detection of chronic inflammation known as elevated neutrophil derived protease activity (EPA) and bacterial pathogenesis known as bacterial protease activity(BPA) in chronic wounds. Despite the evidence, however, clinicians report that incorporating diagnostics into every day practice is challenging and across the globe, they have requested guidance on their use. Methods and Recommendations: A panel of wound care experts, experienced with these tests, met to develop guidelines on their use in wound care practice. The consensus panel concluded that the clinician should test for BPA first. The panel maintained that the risk of invasive infection resulting from the presence of pathogenic bacteria was the greatest threat to the patient\'s health. If the BPA test is negative, the panel recommended testing for EPA. In addition, it was suggested that if the wound failed to progress after the elevated BPA was treated and subsequent testing was negative for BPA, the clinician should consider testing for EPA. Conclusions: In this manuscript, the consensus panel suggests pathways for testing, treating, and retesting for EPA and BPA. The panel expects that following the algorithm has the potential to improve healing outcomes, result in more cost-effective use of advanced therapies, and improve antimicrobial stewardship by guiding antimicrobial use.

Hypervirulent Klebsiella pneumoniae strains have higher potential to cause more severe and disseminated infections than classic K. pneumoniae strains. While initially reported from East Asian countries, cases have now been identified worldwide, sometimes in conjunction with extensive drug resistance. In this issue of the Journal of Clinical Microbiology, T. A. Russo et al. (J Clin Microbiol 56:e00776-18, 2018, https://doi.org/10.1128/JCM.00776-18) validated the diagnostic accuracy of biomarkers that differentiate hypervirulent K. pneumoniae strains from classic strains. This represents a major step forward in building a consensus definition and designing international studies aimed at elucidating the global epidemiology, clinical features, and outcome of this important pathogen.

Similar to Gram-negative organisms, Borrelia spirochetes are dual-membrane organisms with both an inner and outer membrane. Although the outer membrane contains integral membrane proteins, few of the borrelial outer membrane proteins (OMPs) have been identified and characterized to date. Therefore, we utilized a consensus computational network analysis to identify novel borrelial OMPs.
Using a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form β-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes.
Using a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continuously changing. Iceland has a low incidence of MRSA. A \"search and destroy\" policy (screening patients with defined risk factors and attempting eradication in carriers) has been implemented since 1991. Clinical and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the Staphylococcus protein A gene (spa typing), staphylococcal cassette chromosome mec (SCCmec) typing, and screening for the Panton-Valentine leukocidin (PVL) gene. Two hundred twenty-six infected (60%) or colonized (40%) individuals were detected (annual incidence 2.5 to 16/100,000). From 2000 to 2003, two health care-associated outbreaks dominated (spa types t037 and t2802), which were successfully controlled with extensive infection control measures. After 2004, an increasing number of community-associated (CA) cases without relation to the health care system occurred. A great variety of clones (40 PFGE types and 49 spa types) were found, reflecting an influx of MRSA from abroad. The USA300 and Southwest Pacific (SWP) clones were common. SCCmec type IV was most common (72%), and 38% of the isolates were PVL positive. The incidence of MRSA in Iceland has increased since 1999 but remains low and has been stable in the last years. The search and destroy policy was effective to control MRSA in the health care setting. However, MRSA in Iceland is now shifting into the community, challenging the current Icelandic guidelines, which are tailored to the health care system.

Biosynthesis of xanthan polysaccharide, a virulence factor of phytopathogenic Xanthomonas campestris pv. campestris (Xcc), involves the gum operon and the cyclic AMP receptor protein (CRP) homologue Clp. Clp was shown to have the same DNA binding specificity as the CRP at positions 5, 6, and 7 (GTG motif) of the left arm. Therefore, Clp binding sites (CBSs) have typically been identified by pattern searching of the Xcc genome using the consensus CRP binding sequence. Here, results of a reporter assay and electrophoretic mobility shift assay suggest that Clp upregulates the gum operon by binding to two non-consensus sites, in which a more conserved right arm may compensate for the lack of conservation in the left arm, a high GC content in the central region (6 bp) may be important for binding, and binding may be enhanced if the GC-rich central region is palindromic. These suggest that atypical CBSs exist in Xcc promoters and that Clp, while retaining the capacity to bind typical CBSs, has evolved to bind atypical CBS because: 1) Clp shares only moderate homology with the CRP and is modulated by cyclic di-GMP; and 2) Xcc has a higher GC content (65%) than Escherichia coli (50%).

This investigation examines the role of the SaeR/S 2-component system in USA300, a prominent circulating clone of community-associated methicillin-resistant Staphylococcus aureus. Using a saeR/S isogenic deletion mutant of USA300 (USA300DeltasaeR/S) in murine models of sepsis and soft-tissue infection revealed that this sensory system is critical to pathogenesis of USA300 during both superficial and invasive infection. Oligonucleotide microarray and real-time reverse-transcriptase polymerase chain reaction identified numerous extracellular virulence genes that are down-regulated in USA300DeltasaeR/S. Unexpectedly, an up-regulation of mecA and mecR1 corresponded to increased methicillin resistance in USA300DeltasaeR/S. 5\'-RACE analysis defined transcript start sites for sbi, efb, mecA, lukS-PV, hlb, SAUSA300_1975, and hla, to underscore a conserved consensus sequence within promoter regions of genes under strong SaeR/S transcriptional regulation. Electrophoretic mobility shift assay experiments illustrated direct binding of SaeR(His) to promoter regions containing the conserved consensus sequence. Collectively, the findings of this investigation demonstrate that SaeR/S directly interacts with virulence gene promoters to significantly influence USA300 pathogenesis.

BACKGROUND: Computational methods are needed to design multivalent vaccines against flaviviruses (FVs) such as the West Nile virus or the dengue virus (DENV).
OBJECTIVE: We aimed to use physicochemical property (PCP) consensus sequences of FV strains to delineate conserved motifs, areas of maximum variability, and specific loci that correlate with arthropod vector, serotype, and disease severity.
METHODS: PCP consensus sequences for 27 species were prepared from 928 annotated sequences catalogued in Flavitrack. Alignments of these correlated well with the known structures of the NS3 protease domain and envelope (E) proteins. The PCPMer suite was used to identify motifs common to all FVs. Areas of PCP variability that correlated with phenotype were plotted on the structures.
RESULTS: Despite considerable diversity at the amino acid level, PCPs for both proteins were well conserved throughout the FVs. A series of insertions in E separated tick- from mosquito-borne viruses and all arthropod-borne viruses from isolates with no known vector or directly from insects. Comparison of a PCP consensus sequence of E derived from 600 DENV strains (DENV600) with individual ones for DENV1-DENV4 showed that most major serotype-specific variation occurs near these insertions. The DENV600 differed from one prepared from eight hemorrhagic or fatal strains from four DENV serotypes at only three positions, two of which overlap known escape mutant sites.
CONCLUSIONS: Comparing consensus sequences showed that substantial changes occur in only a few areas of the E protein. PCP consensus sequences can contribute to the design of multivalent vaccines.

Certain streptococcal M proteins bind collagen via an octapeptide motif that is located in their hypervariable N-terminal region. The interaction with this extracellular matrix protein enhances adhesion to the host tissue and thereby facilitates infection. Moreover, it has the side effect of eliciting collagen autoimmune responses, a phenomenon which is also observed in patients with acute rheumatic fever. Therefore, the octapeptide motif was named peptide associated with rheumatic fever (PARF). Only a comprehensive characterization of the collagen-binding M proteins and their collagen-binding motifs will allow the investigation of their associations with certain streptococcal infections and their sequelae. Therefore, a collection of Streptococcus dysgalactiae equisimilis strains that were isolated from infected humans was examined, in order to identify collagen-binding proteins and motifs. Strains that bound collagen independent of a hyaluronic acid capsule belonged to 7 distinct types of the emm gene, which codes for the M protein (emm types). Only one of these emm types was previously described as collagen-binding. Five possessed a PARF sequence. The other 2 emm types stC2sk.0 and stG2574 had PARF-like motifs that diverged from the previously described consensus sequence AXYLZZLN but were able to induce collagen autoimmunity when injected into mice. The results led to the amended PARF consensus (A/E/T)XYLXXLN. Moreover, they demonstrate a predictive power regarding collagen-binding and elicitation of collagen autoimmunity, indicating that PARF may be one of the markers for strains that cause collagen-dependent acute rheumatic fever.

virB11, one of the 11 genes of the virB operon, is absolutely required for transport of T-DNA from Agrobacterium tumefaciens into plant cells. Previous studies reported that VirB11 is an ATPase with autophosphorylation activity and localizes to the inner membrane even though the protein does not contain the consensus N-terminal export sequence. In this report, we show that VirB11 localizes to the inner membrane even in the absence of other tumor-inducing (Ti) plasmid-encoded proteins. To facilitate the further characterization of VirB11, we purified this protein from the soluble fraction of an Escherichia coli extract by fusing VirB11 to the maltose-binding protein. The maltose-binding protein-VirB11 fusion was able to complement a virB11 deletion mutant of A. tumefaciens for tumor formation and also localized properly to the inner membrane of A. tumefaciens. The 72-kDa protein, purified from E. coli, exhibited no autophosphorylation, ATPase activity, or ATP-binding activity. To study the importance of the Walker nucleotide-binding site present in VirB11, mutations were generated to replace the conserved lysine residue with either alanine or arginine. Expression of the virB11K175A mutant gene resulted in an avirulent phenotype, and expression of the virB11K175R mutant gene gave rise to an attenuated virulence phenotype. Both mutant proteins were present at levels three to four times higher than that of VirB11 in the wild-type strain. The mutant genes did not exhibit a transdominant phenotype on tumor formation in bacteria that were expressing wild-type virB11. The mutant proteins also localized properly to the inner membrane of A. tumefaciens, but the VirB11K175R protein appeared to be unstable after lysis of the cells.