SUMMARYStrains of bacteria resistant to antibiotics, particularly those that are multiresistant, are an increasing major health care problem around the world. It is now abundantly clear that both Gram-negative and Gram-positive bacteria are able to meet the evolutionary challenge of combating antimicrobial chemotherapy, often by acquiring preexisting resistance determinants from the bacterial gene pool. This is achieved through the concerted activities of mobile genetic elements able to move within or between DNA molecules, which include insertion sequences, transposons, and gene cassettes/integrons, and those that are able to transfer between bacterial cells, such as plasmids and integrative conjugative elements. Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes. This review aims to outline the characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria, focusing on the so-called ESKAPEE group of organisms (, ,, ,, spp., and), which have become the most problematic hospital pathogens.

SUMMARY Staphylococcus aureus bacteremia (SAB) is an important infection with an incidence rate ranging from 20 to 50 cases/100,000 population per year. Between 10% and 30% of these patients will die from SAB. Comparatively, this accounts for a greater number of deaths than for AIDS, tuberculosis, and viral hepatitis combined. Multiple factors influence outcomes for SAB patients. The most consistent predictor of mortality is age, with older patients being twice as likely to die. Except for the presence of comorbidities, the impacts of other host factors, including gender, ethnicity, socioeconomic status, and immune status, are unclear. Pathogen-host interactions, especially the presence of shock and the source of SAB, are strong predictors of outcomes. Although antibiotic resistance may be associated with increased mortality, questions remain as to whether this reflects pathogen-specific factors or poorer responses to antibiotic therapy, namely, vancomycin. Optimal management relies on starting appropriate antibiotics in a timely fashion, resulting in improved outcomes for certain patient subgroups. The roles of surgery and infectious disease consultations require further study. Although the rate of mortality from SAB is declining, it remains high. Future international collaborative studies are required to tease out the relative contributions of various factors to mortality, which would enable the optimization of SAB management and patient outcomes.

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The stable inheritance of genetic material depends on accurate DNA partition. Plasmids serve as tractable model systems to study DNA segregation because they require only a DNA centromere, a centromere-binding protein and a force-generating ATPase. The centromeres of partition (par) systems typically consist of a tandem arrangement of direct repeats. The best-characterized par system contains a centromere-binding protein called ParR and an ATPase called ParM. In the first step of segregation, multiple ParR proteins interact with the centromere repeats to form a large nucleoprotein complex of unknown structure called the segrosome, which binds ParM filaments. pSK41 ParR binds a centromere consisting of multiple 20-base-pair (bp) tandem repeats to mediate both transcription autoregulation and segregation. Here we report the structure of the pSK41 segrosome revealed in the crystal structure of a ParR-DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously to generate the full-length centromere with the ribbon-helix-helix (RHH) fold of ParR binding successive DNA repeats as dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous protein super-helical array, wrapping the DNA about its positive convex surface to form a large segrosome with an open, solenoid-shaped structure, suggesting a mechanism for ParM capture and subsequent plasmid segregation.

Strains of Staphylococcus aureus that are resistant to multiple antimicrobial compounds, including most available classes of antibiotics and some antiseptics, are a major threat to patient care owing to their stubborn intransigence to chemotherapy and disinfection. This reality has stimulated extensive efforts to understand the genetic nature of the determinants encoding antimicrobial resistance, together with the mechanisms by which these determinants evolve over time and are spread within bacterial populations. Such studies have benefited from the application of molecular genetics and in recent years, the sequencing of over a dozen complete staphylococcal genomes. It is now evident that the evolution of multiresistance is driven by the acquisition of discrete preformed antimicrobial resistance genes that are exchanged between organisms via horizontal gene transfer. Nonetheless, chromosomal mutation is the catalyst of novel resistance determinants and is likely to have an enhanced influence with the ongoing introduction of synthetic antibiotics.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.

Increasing evidence within the literature has identified the presence of biofilms in chronic wounds and proposed that they contribute to delayed wound healing. This research aimed to investigate the presence of biofilm in diabetic foot ulcers (DFUs) using microscopy and molecular approaches and define if these are predominantly mono- or multi-species. Secondary objectives were to correlate wound observations against microscopy results in ascertaining if clinical cues are useful in detecting wound biofilm. DFU tissue specimens were obtained from 65 subjects. Scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridisation (PNA-FISH) techniques with confocal laser scanning microscopy (CLSM) were used to visualise biofilm structures. Next-generation DNA sequencing was performed to explore the microbial diversity. Clinical cues that included the presence of slough, excessive exudate, a gel material on the wound bed that reforms quickly following debridement, poor granulation and pyocyanin were correlated to microscopy results. Of the 65 DFU specimens evaluated by microscopy, all were characterised as containing biofilm (100%, P < 0·001). The presence of both mono-species and multi-species biofilms within the same tissue sections were detected, even when DNA sequencing analysis of DFU specimens revealed diverse polymicrobial communities. No clinical correlations were identified to aid clinicians in identifying wound biofilm. Microscopy visualisation, when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and form either mono- or multi-species biofilms. Clinical cues to aid clinicians in detecting wound biofilm are not accurate for use in DFUs. A paradigm shift of managing DFUs needs to consider anti-biofilm strategies.

The concept of biofilms in human health and disease is now widely accepted as cause of chronic infection. Typically, biofilms show remarkable tolerance to many forms of treatments and the host immune response. This has led to vast increase in research to identify new (and sometimes old) anti-biofilm strategies that demonstrate effectiveness against these tolerant phenotypes. Areas covered: Unfortunately, a standardized methodological approach of biofilm models has not been adopted leading to a large disparity between testing conditions. This has made it almost impossible to compare data across multiple laboratories, leaving large gaps in the evidence. Furthermore, many biofilm models testing anti-biofilm strategies aimed at the medical arena have not considered the matter of relevance to an intended application. This may explain why some in vitro models based on methodological designs that do not consider relevance to an intended application fail when applied in vivo at the clinical level. Expert commentary: This review will explore the issues that need to be considered in developing performance standards for anti-biofilm therapeutics and provide a rationale for the need to standardize models/methods that are clinically relevant. We also provide some rational as to why no standards currently exist.