Treatment potential of pathogen-reactive antibodies sequentially purified from pooled human immunoglobulin.

OBJECTIVE: Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with streptococcal and staphylococcal toxic shock, and the Shiga toxin. In addition to this toxin-neutralising activity, IVIG contains other pathogen-reactive antibodies that may confer additional therapeutic benefits. We sought to determine if pathogen-reactive antibodies that promote opsonophagocytosis of different organisms can be sequentially affinity-purified from one IVIG preparation. RESULTS: Antibodies that recognise cell wall antigens of Streptococcus pyogenes, Staphylococcus aureus, and vancomycin-resistant enterococcus (VRE) were sequentially affinity-purified from a single preparation of commercial IVIG and opsonophagocytic activity was assessed using a flow cytometry assay of neutrophil uptake. Non-specific IgG-binding proteins were removed from the S. aureus preparations using an immobilised Fc fragment column, produced using IVIG cleaved with the Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). Affinity-purified anti-S. aureus and anti-VRE immunoglobulin promoted significantly higher levels of opsonophagocytic uptake by human neutrophils than IVIG when identical total antibody concentrations were compared, confirming activity previously shown for affinity-purified anti-S. pyogenes immunoglobulin. The opsonophagocytic activities of anti-S. pyogenes, anti-S. aureus, and anti-VRE antibodies that were sequentially purified from a single IVIG preparation were undiminished compared to antibodies purified from previously unused IVIG.

Virulence profiles of vancomycin-resistant enterococci isolated from surface and ground water utilized by humans in the North West Province, South Africa: a public health perspective.

Vancomycin-resistant enterococci (VRE) have been responsible for numerous outbreaks of serious infections in humans worldwide. Enterococcus faecium and Enterococcus faecalis are the principal species that are frequently associated with vancomycin resistance determinants, thus usually implicated in hospital- and community-acquired infections in humans. The study aim was to determine the antibiotic resistance and virulence profiles of VREs isolated from surface and groundwater samples that are used by humans in the North West Province, South Africa. A total of 170 water samples were collected and analyzed. Eighty-one potential isolates were screened for characteristics of Enterococcus species using preliminary biochemical tests, PCR assays and sequence analysis. The antimicrobial resistance profiles of the isolates against nine antibiotics were determined and a dendrogram was generated to access the relatedness of the isolates. The isolates were screened for the presence of antibiotic resistance and virulence genes by multiplex PCR analysis. A total of 56 isolates were confirmed as Enterococcus species and the proportion of E. faecium (46.9%) was higher than E. faecalis (29%) and E. saccharolyticus (1.2%). Sequence data of E. faecium, E. faecalis, and E. saccharolyticus isolates revealed 97 to 98% similarities to clinical strains deposited in NCBI Genbank. Large proportions (44; 78.6%) of the isolates were resistant to vancomycin while 16 and 3.6% of the isolates possessed the vanA and vanB genes respectively. The MAR phenotype Vancomycin-Nalidixic Acid-Streptomycin-Chloramphenicol-Ampicillin-Oxytetracycline-Gentamycin-Nitrofurantoin-Sulphamethoxazole indicated that some isolates were resistant to all of the nine antibiotics tested. Cluster analysis of antibiotic resistance data revealed two major clusters. Sixteen (36.4%), 14 (27.3%), 3 (6.8%), and 2 (4.5%) of the VRE isolates possessed the gel, asa1, hyl, and esp virulence genes respectively while the cylA gene was not detected in the study. Multiple antibiotic-resistant enterococci were also resistant to vancomycin and possessed virulence determinants indicating that they can pose severe public health complications on individuals who consume contaminated water.

A new structural class of bacterial thioester domains reveals a slipknot topology.

An increasing number of surface-associated proteins identified in Gram-positive bacteria are characterized by intramolecular cross-links in structurally conserved thioester, isopeptide, and ester domains (TIE proteins). Two classes of thioester domains (TEDs) have been predicted based on sequence with, to date, only representatives of Class I structurally characterized. Here, we present crystal structures of three Class II TEDs from Bacillus anthracis, vancomycin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium. These proteins are structurally distinct from Class I TEDs due to a ß-sandwich domain that is inserted into the conserved TED fold to form a slipknot structure. Further, the B. anthracis TED domain is presented in the context of a full-length sortase-anchored protein structure (BaTIE). This provides insight into the three-dimensional arrangement of TIE proteins, which emerge as very abundant putative adhesins of Gram-positive bacteria.

MALDI-TOF MS meets WGS in a VRE outbreak investigation.

The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.