Genomic analysis revealed distinct transmission clusters of vancomycin-resistant Enterococcus faecium ST80 in Stockholm, Sweden.

Vancomycin-resistant Enterococcus faecium (VREfm) belonging to sequence type (ST)80 has become the predominant clonal lineage in Stockholm in the last three years. ST80 accounted for 75% and 46% of VRE cases in 2018 and 2019, respectively, and gave rise to both vanA-type and vanB-type outbreaks. Non-duplicate ST80-VREfm isolates (N = 188) were subjected to whole genome sequencing. Genomic analysis revealed three distinct transmission clusters. Our study indicated that difficulties in detecting low-grade vancomycin-resistant isolates by phenotypic testing might be one of the explanatory factors for the prolonged course of vanB-type outbreaks. Herein, we also report the first optrA-positive linezolid-resistant VRE isolate in Stockholm.

Transmission events and antimicrobial susceptibilities of methicillin-resistant Staphylococcus argenteus in Stockholm.

OBJECTIVES: Staphylococcus argenteus has been increasingly reported since the species was defined as a novel staphylococcal species in 2015. This study aims to investigate genetic epidemiological links and antimicrobial susceptibilities of methicillin-resistant S. argenteus isolates recovered in Stockholm. METHODS: Sixteen methicillin-resistant S. argenteus isolates were identified from a collection of methicillin-resistant Staphylococcus aureus in Stockholm 2007-2018, by using whole-genome sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The genomes of the isolates were investigated by pulsed-field gel electrophoresis, single-nucleotide polymorphism (SNP)-based phylogeny, k-mer analysis, core-genome multi-locus sequence typing (cgMLST), resistance traits and virulence factors. The MICs of 19 antimicrobial agents for each isolate were determined by using the broth microdilution method. RESULTS: Of the 16 isolates, seven, seven and two isolates were assigned to ST1223, ST2250 and ST2793, respectively, with the S. aureus MLST-scheme. Analyses based on SNPs and cgMLST revealed a likely clonal spread of methicillin-resistant S. argenteus in 2007. Four isolates were found to be resistant to non-ß-lactams in antimicrobial susceptibility testing. CONCLUSIONS: A transmission event of methicillin-resistant S. argenteus in family was identified by this study. Among our limited number of isolates, non-ß-lactam resistance was detected, which highlights the necessity of a continued surveillance on this emerging pathogen. S. argenteus could be correctly identified by MALDI-TOF MS with the updated database, enabling its detection also in clinical laboratories.

Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates.

The purpose of this investigation was to compare the performance of species-specific polymerase chain reaction (PCR), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for the identification of Enterococcus species. A total of 132 clinical isolates were investigated by the following: (1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium, ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS; (3) VITEK MS; and (4) the VITEK 2 system. 16S rRNA gene sequencing was used as a reference method in the study. The 132 isolates were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and 21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly at the species level. The VITEK 2 system could identify 131/132 (99.2 %) and 121/132 (91.7 %) of the isolates at the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and 81.3 % (13/16) of the E. casseliflavus isolates. The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF MS is a rapid, reliable and cost-effective identification technique for enterococci. The VITEK 2 system is less efficient at detecting non-faecalis and non-faecium Enterococcus species.

Screening for vancomycin-resistant enterococci: an efficient and economical laboratory-developed test.

A laboratory-developed test (Lab Assay), combining enrichment broth and real-time polymerase chain reaction (PCR) for vancomycin-resistant enterococci (VRE) screening, was developed and evaluated in this study. A total of 1,765 faecal or rectal swabs sent to the laboratory for VRE screening were investigated in parallel by Lab Assay and the Roche LightCycler VRE detection kit-based method. The diagnostic values for Lab Assay were as follows: 100% sensitivity, 79.92% specificity, 1.94% positive predictive value and 100% negative predictive value, which were comparable to the results from the LightCycler kit-based assay. The detection limit of Lab Assay was 10(0) to 10(1) colony-forming units (CFU)/ml of inoculum in broth for both VanA-type and VanB-type VRE. The PCR method developed in this study was approved to be applicable on both the Applied Biosystems 7500 Fast Real-Time PCR System and the LightCycler(®) 480 Real-Time PCR System. The flexibility in choosing PCR systems makes it possible that the PCR assay could be fully compatible with the DNA extraction's platform, providing an integrated workflow. Furthermore, the material cost is saved at 7EUR per sample when Lab Assay replaces the commercial kit-based method in our routine screening for VRE. Therefore, the laboratory-developed broth-PCR method is an efficient and economical assay for VRE screening.

Surface bound plasmin promotes migration of Streptococcus pneumoniae through reconstituted basement membranes.

Penetration of basement membrane is believed to be an essential step in the pathogenesis of bacterial meningitis. Consequently Streptococcus pneumoniae strains were tested for their ability to adhere to reconstituted basement membrane (Matrigel) and in a proceeding step penetrate this membrane by the use of surface activated plasmin. A majority of S. pneumoniae strains tested were found to adhere to reconstituted basement membrane as well as to the purified laminin component. Three out of seventeen strains also adhered to the collagen IV component. All the investigated strains also demonstrated a capacity to bind plasminogen with up to 42,000 plasminogen binding sites per bacterium as estimated by Scatchard analysis. Two strains selected for optimal adhesion and plasminogen binding were further tested for basement membrane penetration using a dual chamber model. Our data show that penetration was achieved within 3-4 h in the presence of plasminogen whereas without plasminogen no strain was able to penetrate during a 21 h incubation. The results suggest a potential role of surface associated plasminogen in bacterial penetration of basement membranes and extracellular matrix.

Binding to human extracellular matrix by Neisseria meningitidis.

Adhesion of Neisseria meningitidis strains to extracellular matrix (ECM) and purified matrix components was examined. Most strains bound to subendothelial ECM as well as to immobilized fibronectin and types I, III, and V collagen. Strains from healthy carriers adhered significantly better than isolates from patients. The binding site was localized to the central 75-kDa cell-binding domain of the fibronectin molecule. This domain has not been described previously to interact with bacterial structures.

Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.

Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.

Interaction of Haemophilus influenzae with the mammalian extracellular matrix.

The adhesiveness of 2 unencapsulated nonfimbriated strains of Haemophilus influenzae, 23459 and 23330, and the encapsulated fimbriated strain 770235 to extracellular matrix (ECM) and to its isolated components was studied, as was the potential of H. influenzae plasminogen receptors to enhance degradation of ECM and bacterial penetration through basement membrane. All strains exhibited efficient adhesiveness to reconstituted basement membrane and to ECM from cultured human endothelial cells. Strains 23459 and 23330 efficiently adhered to immobilized laminin, fibronectin, and various collagens. Strain 770235 adhered efficiently to fibronectin and type I and III collagens and with low efficiency to laminin. With all 3 strains, plasmin generated on H. influenzae plasminogen receptors degraded laminin and fibronectin as well as ECM from human endothelial cells. Plasmin bound on H. influenzae cells also potentiated penetration of bacteria through a basement membrane preparation reconstituted on membrane filters. These results give evidence for a role of ECM adherence and plasminogen activation in the spread of H. influenzae through tissue barriers.

Binding of tissue-type plasminogen activator (t-PA) to Neisseria meningitidis and Haemophilus influenzae.

Forty-nine bacterial strains representing five species known to interact with human plasminogen were tested for the ability to bind the two major human plasminogen activators, t-PA and urokinase. The bacterial species tested included Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus equisimilis and human group G streptococci. All N. meningitidis and 11 of 14 H. influenzae strains displayed substantial binding of t-PA with values in the range of 20-46%. On the contrary, none of the streptococcal strains bound significant amounts of tPA. With urokinase no binding could be found for any of the bacterial species tested. Scatchard analysis with a selected H. influenzae strain (HI23354) demonstrated 10,000 receptors per bacterium for t-PA with a Kd value of about 20 nmol l-1. The corresponding values with a selected N. meningitidis strain (Mo 52) was 8500 receptors per bacterium and 70 nmol l-1. t-PA binding could be reduced about 40% by the addition of 10 mmol l-1 of the lysine analogue epsilon-aminocaproic acd (EACA) whereas no inhibitory effect could be demonstrated with arginine. Addition of 2 mumol l-1 of plasminogen which is enough to occupy all bacterial sites for plasminogen did not interfere with the t-PA binding, suggesting that the receptors for t-PA and plasminogen are distinct. Using very high plasminogen concentrations however, t-PA binding could be reduced by about 50% possibly due to an interaction between t-PA and plasminogen in the fluid phase. Our results demonstrate the occurrence of a previously unknown type of bacterial receptor that is capable of specifically binding t-PA.

Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoeae and formation of surface-associated plasmin.

Forty-two strains of Neisseria meningitidis and 17 of Neisseria gonorrhoeae were tested for their ability to interact with 125I-labeled Glu-plasminogen. All strains tested reacted substantially with plasminogen, resulting in uptake values of 20%-48%. Scatchard analysis with selected N. meningitidis strains demonstrated a dual-phase receptor interaction, one more avid receptor with a Kd of 50 nM and 3000-6000 receptors per bacterium and a second receptor with a Kd of 200 nM and 10,000-20,000 receptors per bacterium. Plasminogen uptake could be completely eliminated by low concentrations of epsilon-aminocaproic acid, suggesting that the lysine binding sites on the plasminogen molecule are involved in the receptor-ligand interaction. The binding of plasminogen to the bacterial receptor facilitates the tissue-type plasminogen activator-mediated conversion to Glu-plasmin, which also modifies itself to the Lys form. Receptor-associated plasmin is enzymatically active, monitored as a breakdown of the chromogenic substrate S-2251, and retains its activity in the presence of naturally occurring inhibitors in plasma.

Tissue-type plasminogen activator-mediated activation of plasminogen on the surface of group A, C, and G streptococci.

The interaction of Glu-plasminogen with group A, C, and G streptococci and subsequent formation of surface-associated plasminogen by tissue-type plasminogen activator (t-PA) were studied. Binding of 125I-Glu-plasminogen to streptococci greatly facilitated its activation to 125I-Glu-plasmin by exogenous t-PA, whereas activation in the absence of bacteria took place only slowly. Glu-plasmin formed on the streptococcal surface was further converted to the Lys form. Similar activation and modification took place also in the presence of plasminogen-depleted plasma, containing functional t-PA and plasmin inhibitors, indicating that the surface-associated enzymes were protected against these inhibitors. Lys-plasminogen was 10- to 30-fold more potent than Glu-plasminogen or Glu-plasmin in inhibiting the binding of 125I-Glu-plasminogen to streptococci. This indicated a higher affinity of the Lys form towards plasminogen-binding molecule(s) on the streptococcal surface. The surface-associated plasmin was also enzymically active as judged by digestion of chromogenic substrate S-2251. Surface-associated plasmin activity was observed only when the incubations were carried out in the presence of t-PA and Glu-plasminogen or human plasma as the source of plasminogen. Under these conditions, soluble enzymatic activity was also recovered in the supernatant of group A streptococci. This favors the idea that plasmin can be released from the bacterial surface. The findings provide a mechanism for streptococci to adopt proteolytic activity by binding a host-derived enzyme zymogen on their surface, where the subsequent activation then takes place. The results suggest a role for surface-associated plasmin activity in tissue tropism and tissue invasiveness of streptococci.

Two types of receptors for human plasminogen on group G streptococci.

To investigate the nature of plasminogen binding to streptococci, strains selected for high reactivity with human plasminogen were examined for binding pattern against a panel of plasminogen fragments. The strains included human isolates of groups A, C and G as well as bovine isolates of group G. All strains reacted substantially with the plasminogen fragment kringle 1-3. Using the miniplasminogen fragment (kringle 5 and the B chain) a small but reproducible uptake was detected for human group G strains but not for group A or C strains. The group G strains of bovine origin on the other hand demonstrated high uptake of miniplasminogen, suggesting the possibility of an alternative plasminogen receptor for this species. This interpretation was supported by blocking experiments with the lysine analogue EACA where low concentrations (1 mM) completely blocked plasminogen binding to human streptococci, whereas a 100-fold higher concentration was needed for bovine group G strains. Scatchard plots with human isolates resulted in straight lines and Kd values were generally in the range of 20-80 nM. The number of receptors was estimated to be 45,000 for a selected group A strain and about 10,000 for the selected group C and G strains. Scatchard analysis with bovine group G isolates on the other hand revealed a two phase interaction, supporting the assumption of two different receptor structures on these strains. Kd for the first phase was estimated to be about 20 nM (10,000-20,000 receptors per bacterium), which was similar to the human strains, whereas the second phase was in the range of 400-500 nM (50,000 and 150,000 receptors per bacterium with two selected strains). Scatchard plots with the miniplasminogen fragment as ligand mimicked the phase two reaction with plasminogen, supporting the concept that this reaction represents a new and not previously described receptor. Both the receptor reacting with the kringle 1-3 portion and the one reacting with the miniplasminogen portion bound plasmin and plasminogen with similar affinity.

Surface-associated activation of plasminogen on gram-positive bacteria. Effect of plasmin on the adherence of Staphylococcus aureus.

In this article we review a novel type of plasminogen activation on staphylococcal and streptococcal cells. The activation mechanism implies a specific binding of glu-plasminogen to bacterial surface via the lysine-binding sites of plasminogen. Association of plasminogen with bacterial surfaces greatly enhances the t-PA mediated activation which takes place only poorly in solution. The end product, surface-associated plasmin, is enzymatically active, protected against high molecular weight plasmin inhibitors and capable of converting itself from glu-plasmin to the lys-form. The modification is associated with an increased affinity of the bound lys-plasmin towards the binding molecules on bacterial surface. This novel way of retaining plasmin on the surface may be important for the bacteria to invade and penetrate surrounding tissues. Our data on the effect of plasmin on staphylococcal adherence indicate that plasmin is not very effective in cleaning bacteria from surfaces coated with extracellular matrix components, fibronectin and fibrinogen.

Receptors for human plasminogen on the biological response modifier OK-432.

The biological response modifier OK-432, constituting cell wall fragments from a group A Streptococcus strain and used in anticancer therapy trials, was tested for its ability to interact with different plasma proteins. The uptake of 125I-labelled protein was measured using a panel of six different plasma proteins all known to react with receptors on a majority of streptococcal strains. Of the proteins tested, plasminogen demonstrated the most substantial uptake, with uptake values ranging from 70 to 79%. A slight interaction with fibrinogen was also detected whereas no significant interaction was found with either human immunoglobulin (Ig)A, IgG, serum albumin, or mouse albumin. The results with plasminogen suggest the possibility of a new explanation of the antitumor activity described for OK-432.

Generation of human LAK cells in tissue culture bags using recombinant IL-2 and serum free medium. Effects of pretreatment with phenylalanine-methylester.

A technique for processing and culturing of human LAK cells using an automated closed system and tissue culture bags is described. To circumvent the inhibitory effects of monocytes on LAK cells the peripheral blood mononuclear cells (PBMC) were pretreated with phenylalanine-methylester (PheOMe). PBMC were obtained from healthy donors by leukapheresis of whole blood. After pretreatment with PheOMe and culturing with IL-2 for 96 h, 60% of the cells remained. PheOMe significantly reduced the number of monocytes (Leu-M3+ cells) from 20-12%. The lytic activity (against K562 and Daudi) of non-PheOMe-treated cells reached a plateau at 72-96 h while PheOMe-treated cells reached maximum activity at 96 h. The total lytic activity/tissue culture bag at 96 h of PheOMe-pretreated cells was significantly augmented in comparison to non-PheOMe-pretreated cells. The present technique allows rapid and simple generation of LAK cells without serum in sterile receptacles suitable for therapy. Additionally, the LAK cell efficacy was improved by reducing the inhibitory effects of monocytes.

Receptors for human plasminogen on gram-negative bacteria.

A total of 188 strains representing 11 species of gram-negative bacteria were examined for the ability to interact with human plasminogen. Highly purified human plasminogen was labeled with 125I, and its uptake by different bacterial strains was measured. All 14 strains of Haemophilus influenzae and all 13 strains of Branhamella catarrhalis tested were positive with respect to plasminogen uptake. Also, eight species belonging to the family Enterobacteriaceae were tested, and of those, Proteus mirabilis demonstrated the most substantial uptake, with 28 of 39 strains taking up more than 10% of the plasminogen. Ten strains of Pseudomonas aeruginosa were also tested, of which seven showed uptake values higher than 10%. With H. influenzae and B. catarrhalis strains, Scatchard analysis indicated a two-phase receptor interaction, one more-avid receptor with a Kd of 6 to 8 nM and 2,000 to 2,500 sites per bacterium and a second receptor with a Kd of 50 to 80 nM and 9,000 sites per bacterium. With Pseudomonas aeruginosa strains, a single receptor interaction was detected with a Kd of 60 nM and the number of sites was estimated as 8,000 per bacterium. Scatchard analysis with strains of P. mirabilis indicated binding of a less-specific nature. However, plasminogen uptake by this species could be reduced by 50% by the addition of 2 mM unlabeled plasminogen. This estimate of Kd, as well as uptake studies with plasminogen fragments, suggests different properties of this receptor. With all receptor types, the addition of plasmin-aprotinin complex inhibited plasminogen uptake, which demonstrates that both forms of the molecule react with the same receptors. Plasminogen uptake could be eliminated by the addition of lysine or epsilon-aminocaproic acid, which suggests that the lysine-binding sites of the plasminogen molecule are involved in the receptor-ligand interaction.

New receptor for human plasminogen on gram positive cocci.

180 bacterial strains representing 17 different species of gram positive cocci were tested for the ability to interact with human plasminogen. Receptors for plasminogen could be detected on 23/24 strains of S. pyogenes, 15/15 strains of S. equisimilis, 14/16 strains of human group G streptococci and 14/14 strains of S. pneumoniae. Eight of nineteen strains representing five species of alpha-hemolytic streptococci were also positive. S. equisimilis demonstrated the highest uptake with a median value of 58 per cent (20%-67%). On the other hand, all strains of S. agalactiae, the majority of S. faecalis and all S. aureus, S. epidermidis and S. saprophyticus strains tested were negative. The concentration of unlabelled plasminogen causing a 50 per cent reduction of bound tracer was between 50 and 150 mM. These estimates of the dissociation constant confirmed the specific nature of the interaction. Binding of plasminogen could be blocked by addition of plasmin-aprotinin complex, suggesting that plasminogen and plasmin bind to the same receptor. Binding was also blocked by the plasminogen fragment kringle 1-3, but not by miniplasminogen, a fragment containing kringle 5 and the B-chain region. As streptokinase interacts mainly with the B-chain of plasmin it is clear that the bacterial receptor for plasminogen is not identical to streptokinase.

Antibody- and interferon-dependent killer cells are part of the NK cell receptor positive subpopulation of human peripheral blood cells.

Cytotoxicity by human non-adherent peripheral blood lymphocytes was analysed after effector cell activation with either interferon (IF) or by target cell specific IgG antibodies (T-IgG). Four different cell lines were used as target cells that differed in susceptibility to natural killer cell (NK) activity; a highly susceptible T cell line (Molt-4), a medium susceptible B lymphoma line (Daudi), a resistant B cell line established by Epstein-Barr virus transformation (LCL-LS) and a resistant mouse mastocytoma line (P815). Three different parameters influencing killing were investigated; lytic potential, target cell binding and efficiency of the lytic phase from which the absolute number of effector cells and their recycling capacity could be estimated. It was found that, when using human target cell lines, IF and T-IgG influenced the system in a similar way by activating the lytic phase and the effector cell recycling but not the early binding phase. With the NK resistant mouse mastocytoma cell line P815 a comparatively small target binding population was found which, however, increased markedly with T-IgG treatment. Taken together, the results indicate that the effector population responsible for antibody-induced killing belong to a subpopulation of cells that have the ability to spontaneously conjugate to the present target cells by virtue of naturally occuring undefined cell surface receptors designated NKR (NK receptor) and that the role of T-IgG in the present system is similar to that of IF. In contrast, if target cells are used that do not express binding structures for NKR receptors, T-IgG may also fulfill a receptor function through Fc receptors for IgG.

Inhibition of human NK cell cytotoxicity by induction of cyclic AMP depends on impaired target cell recognition.

Induction of cyclic AMP (cAMP) depresses natural killer (NK) cell activity. The present results demonstrate that this is dependent on a decreased capacity of the effector cells to conjugate to target cells. This was found either if dibutyryl-cAMP was used or if cAMP was induced by adenylate cyclase stimulation with prostaglandin E1 (PGE1) or by inhibition of phosphodiesterase activity with the inhibitor ZK 62711. The sites of action for cAMP-induced NK suppression and interferon (IFN)-induced NK enhancement are demonstrated to be distinct, since IFN acts by increasing the lytic efficiency and the recycling capacity without influencing target binding. Sequential treatment with cAMP/IFN and IFN/cAMP shows that IFN can neither restore target binding when added after cAMP nor protect against the cAMP-induced target binding inhibition when added before cAMP. The results are discussed in view of earlier data on cAMP in relation to cell membrane functions and cellular recognition, the mechanism underlying the cAMP-induced target binding inhibition, and the potential of the NK system as an indicator for immunosuppression. The present work also demonstrates the particular subpopulation in peripheral blood which mediates most NK activity, to respond strongly to PGE1 stimulation with regard to cAMP induction.