Relationship between endotoxin core, staphylococcal and varicella antibody levels and outcome following aortic valve replacement surgery: a prospective observational study.

BACKGROUND: Morbidity and mortality following cardiac valve surgery is high. Immunity is an important contributor to outcome. This study examines the relationship of staphylococcal and endotoxin antibody levels to outcome following cardiac surgery. METHODS: Using enzyme-linked immunosorbent assays (ELISA), we measured pre-operative levels of antibodies to endotoxin core (EndoCAb); 3 common staphylococcal epitopes and varicella on saved serum of 60 adult patients scheduled to undergo elective primary surgical aortic valve replacement (AVR). Primary outcome measure was post-operative length of stay (LOS) in hospital with secondary outcomes being development of infective complications, length of stay on the intensive care unit (ICU) and 30-day mortality. Patients were quartiled according to antibody levels and outcomes compared between the quartile groups using Mann-Whitney tests for length of stay and Fisher's test for development of infection. RESULTS: Sixty patients (34 M, 26 F) were recruited with mean age 73 years (IQR 66-78), mean body mass index (BMI) 27.7 (IQR 25-31) and EuroSCORE II 1.44 (0.95-1.99). Those patients in the lower quartile for pre-operative antibody level had a longer post-operative stay than the upper quartile. EndoCAb (median IgG level Q1 42.2 MU/ml vs Q4 256 MU/ml) 9 vs 6 days, p = 0.025; alpha-toxin (median IgG level Q1 63 U vs Q4 558 U) 10 vs 7 days, p = 0.034; teichoic acid (median IgG level Q1 14 U vs Q4 419 U) 10 vs 8 days, p = 0.441; staphylococcal enterotoxin A (median IgG level Q1 55 U vs Q4 427 U) 9 vs 7 days, p = 0.865; varicella zoster (median IgG level Q1 1.325 U vs Q4 2.54 U) 8 vs 7 days, p = 1.0; and combined antibody levels 10 vs 6 days, p = 0.017. There were no differences in the number developing post-operative infections for each antibody type. The combined antibody analysis suggested a reduction in proportion of individuals developing infection from the upper vs lower quartile: 0 vs 0.33, p = 0.042. CONCLUSIONS: This study again suggests the inverse relationship between endotoxin core antibody levels and outcome following aortic valve surgery as well as suggesting a similar relationship with antibodies to staphylococcus. There is no such relationship for antibody levels against an organism not providing a peri-operative threat. Understanding this relationship may enable therapeutic manipulation of immune status, re-evaluation of risk and further investigation of the low immune state. TRIAL REGISTRATION: The patients in this study are a sub-group of the RELIEF AS study.ClinicalTrials.gov identifier NCT02174471.

New Insights into the Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus Host Interaction Mechanisms.

Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) were classically clustered into the Lancefield Group D streptococci and despite their taxonomic reclassification still share a similar genetic content and environment. Both species are considered as opportunistic pathogens. E. faecium is often associated with nosocomial bacteraemia, and S. gallolyticus is sporadically found in endocarditis of colorectal cancer patients. In both cases, the source of infection is commonly endogenous with a translocation process that launches through the intestinal barrier. To get new insights into the pathological processes preceding infection development of both organisms, we used an in vitro model with Caco-2 cells to study and compare the adhesion, invasion and translocation inherent abilities of 6 E. faecium and 4 S. gallolyticus well-characterized isolates. Additionally, biofilm formation on polystyrene, collagen I and IV was also explored. Overall results showed that E. faecium translocated more efficiently than S. gallolyticus, inducing a destabilization of the intestinal monolayer. Isolates Efm106, Efm121 and Efm113 (p < .001 compared to Ef222) exhibited the higher translocation ability and were able to adhere 2-3 times higher than S. gallolyticus isolates. Both species preferred the collagen IV coated surfaces to form biofilm but the S. gallolyticus structures were more compact (p = .01). These results may support a relationship between biofilm formation and vegetation establishment in S. gallolyticus endocarditis, whereas the high translocation ability of E. faecium high-risk clones might partially explain the increasing number of bacteraemia.

Increase of faecal tryptic activity relates to changes in the intestinal microbiome: analysis of Crohn's disease with a multidisciplinary platform.

OBJECTIVE: To investigate-by molecular, classical and functional methods-the microbiota in biopsies and faeces from patients with active Crohn's disease (CD) and controls. DESIGN: The microbiota in biopsies was investigated utilizing a novel molecular method and classical cultivation technology. Faecal samples were investigated by classical technology and four functional methods, reflecting alterations in short chain fatty acids pattern, conversion of cholesterol and bilirubin and inactivation of trypsin. RESULTS: By molecular methods we found more than 92% similarity in the microbiota on the biopsies from the two groups. However, 4.6% of microbes found in controls were lacking in CD patients. Furthermore, NotI representation libraries demonstrate two different clusters representing CD patients and controls, respectively. Utilizing conventional technology, Bacteroides (alt. Parabacteroides) was less frequently detected in the biopsies from CD patients than from controls. A similar reduction in the number of Bacteroides was found in faecal samples. Bacteroides is the only group of bacteria known to be able to inactivate pancreatic trypsin. Faecal tryptic activity was high in CD patients, and inversely correlated to the levels of Bacteroides. CONCLUSIONS: CD patients have compositional and functional alterations in their intestinal microbiota, in line with the global description hypothesis rather than the candidate microorganism theory. The most striking functional difference was high amount of faecal tryptic activity in CD patients, inversely correlated to the levels of Bacteroides in faeces.

The Staphylococcus aureus alpha-toxin perturbs the barrier function in Caco-2 epithelial cell monolayers by altering junctional integrity.

Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca(2+)](i)), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood.

Biological relevance of natural alpha-toxin fragments from Staphylococcus aureus.

Serine proteases represent an essential part of cellular homeostasis by generating biologically active peptides. In bacteria, proteolysis serves two different roles: a major housekeeping function and the destruction of foreign or target cell proteins, thereby promoting bacterial invasion. In the process, other virulence factors such as exotoxins become affected. In Staphylococcus aureus culture supernatant, the pore-forming alpha-toxin is cleaved by the coexpressed V8 protease and aureolysin. The oligomerizing and pore-forming abilities of five such spontaneously occurring N- and C-terminal alpha-toxin fragments were studied. (3)H-marked alpha-toxin fragments bound to rabbit erythrocyte membranes but only fragments with intact C termini, missing 8, 12 and 71 amino acids from their N-terminal, formed stable oligomers. All isolated fragments induced intoxication of mouse adrenocortical Y1 cells in vitro, though the nature of membrane damage for a fragment, degraded at its C terminus, remained obscure. Only one fragment, missing the first eight N-terminal amino acids, induced irreversible intoxication of Y1 cells in the same manner as the intact toxin. Four of the isolated fragments caused swelling, indicating altered channel formation. Fragments missing 12 and 71 amino acids from the N terminus occupied the same binding sites on Y1 cell membranes, though they inhibited membrane damage caused by intact toxin. In conclusion, N-terminal deletions up to 71 amino acids are tolerated, though the kinetics of channel formation and the channel's properties are altered. In contrast, digestion at the C terminus results in nonfunctional species.

Caracterización fenotípica y genotípica de la resistencia a meticilina de cepas de Staphylococcus aureus aisladas en Antofagasta / Phenotypic and genotypic characterization of methicillin resistance in strains of Staphylococcus aureus isolated in Antofagasta

Staphylococcus aureus resistant to methicillin (SARM) has been associated with nosocomial infections due to its capacity to develop resistance to multiple antibiotics. There is little information about the SARM which are found in the hospital services of Antofagasta. We studied the phenotypic and genotypic characteristics of methicillin resistance in 38 strains of S. aureus isolated in Antofagasta, identified by coagulase and API Staph tests and by a biochemical test (Ph-system). The susceptibility to antibiotics was studied using the agar dilution technique, identifying SARM strains with discs of oxacillin. Beta-lactamase with nitrocephine, and the gene mecA by means of PCR. Eighty nine percent (34 strains) were SARM with a high resistance to ampicillin, penicillin, erythromycin, claritromycin. gentiamycin, amikacine and ciprofloxacine. All isolates were susceptible to vancomycin and rifampicin. Beta-lactamase was demonstrated in 79% of the SARM strains. Strain typing and resistance patterns revealed a great diversity of PhP-types and antibiotypes in the isolates. Ninety seven percent of the SARM strains had the gene mecA. One PhP-type (C6) was dominant (5 SARM strains) all had the mecA gene, produced beta lactamase and had the same pattern of antibiotic resistance. We conclude that the dominant phenotypes of SARM strains which have the mecA gene and multiple resistance to antibiotics are present in the hospitals of Antofagasta, and sound the alert on the risk of nosocomial transmission of epidemic clones of SARM.

Staphylococcus aureus resistentes a meticilina (SARM) han sido asociados con infecciones nosocomiales por su capacidad para desarrollar resistencia a múltiple antibióticos, existiendo escasa información acerca de SARM que están circulando en los servicios hospitalarios de Antofagasta. Se estudió características fenotIpicas y genotípicas de la resistencia a meticilina en 38 cepas de S. aureus aisladas en Antofagasta, identificadas por tests de coagulasa y API Staph y por tipificación bioquímica (Ph-Sistem). La susceptibilidad a antibióticos se realizo por técnica de dilución en agar, las cepas SARM fueron identificadas con discos de oxacilina, beta-lactamasa por nitrocefina y gen mecA fue detectado pot PCR. El 89% (34 cepas), fueron SARM con una alta resistencia a ampicilina, penicilina, eritromicina, gentamicina, amikacina y ciprofloxacino. Todos los aislados fueron susceptibles a vancomocina y rifampicina. Beta lactamasa fue demostrada en 79% de las cepas SARM. La tipificación y los patrones de resistencia revelaron una alta diversidad de PhP tipos y antibioticos en los aislamientos. El 97% de las cepas SARM albergaban el gen mecA. Un PhP tipo (C6) fue dominante. (5 cepas SARM), todos presentando el gen mecA, produciendo beta lactamasa y mostrando el mismo patrón de resistencia antibiótica. Se concluye que los fenotipos dominantes de cepas SARM que albergan el gen mecA y resistencia múltiples alos antibióticos están circulando en los hospitales de Antofagasta, alertando sobre el riesgo de transmisión intranosocomial de clones epidémicos de SARM.

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract.

BACKGROUND: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. RESULTS: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. CONCLUSION: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model.

Prevalencia de Enterococos resistentes a antibióticos en aguas servidas en el norte de Chile / Prevalence of antibiotic resistant Enterococcus spp in waste waters in the north of Chile

Background: There is little information available in Chile on the distribution of Enterococcus spp in waste water and its implications in transmission of antibiotic resistance through the water cycle. Enterococcus spp are common in nosocomial infections and may spread antibiotic resistance through the food chain. Aim: To determine the presence of antibiotic resistant Enterococcus spp in the sewage of Antofagasta, Chile. Material and Methods: Samples of sewage from two sewage treatment plants and from the Public Hospital of Antofagasta collector were obtained. Enterococcus spp were isolated on m-Enterococcus agar containing ampicillin, vancomycin and streptomycin. The isolates were identified and subjected to biochemical typing (PhPlate). Minimal inhibitory concentration determination was performed by agar dilution technique. Results: High counts of resistant Enterococcus spp were found on the streptomycin plates, lower on ampicillin and very low on vancomycin plates. A total of 63 Enterococcus spp strains were typed and the identification showed 5 different species; E faecalis (65%), E faecium (14%), E hirae (13%), E durans (6%) and E gallinarum (2%). The typing revealed a high diversity among the isolates. Two biochemical phenotypes were predominant, C1 (21 strains) and C6 (7 strains). Both were highly resistant to gentamycin and streptomycin; moderately resistant to ampicillin, cloramphenicol, tetracycline and ciprofloxacin, and with intermediate susceptibility to vancomycin. Both phenotypes were found in the sewage of the hospital collector and in the treatment plants. Conclusions: In the sewage of Antofagasta we found dominating phenotypes of multiresistant Enterococcus spp. Sewage could be an important way of transmission of these microorganisms.

An anthroposophic lifestyle and intestinal microflora in infancy.

The intestinal flora is considered to have an impact on the development of the immune system. In the anthroposophic lifestyle, a diet comprising vegetables spontaneously fermented by lactobacilli, and a restrictive use of antibiotics, anti-pyretics and vaccinations, is typical. The aim of this study was to assess the gut flora in infants in relation to certain lifestyle characteristics associated with anthroposophy. Sixty-nine children < 2 years of age with an anthroposophic lifestyle, and 59 infants of a similar age with a traditional lifestyle, were clinically examined and questionnaire replies assessed. Fecal samples were analyzed by bacterial enumeration, bacterial typing through biochemical fingerprinting and by measuring microflora-associated characteristics (MACs). The numbers of colony-forming units (CFU)/g of feces were significantly higher for enterococci and lactic acid bacteria in children who had never been exposed to antibiotics (5.5 x 107 vs. 2.1 x 107; p < 0.001 and 10 x 107 vs. 4.1 x 107; p < 0.01, respectively). Furthermore, the number of enterococci was significantly higher in breastfed and vegetarian infants (p < 0.01). The diversity (Simpson's diversity index) of lactobacilli, as determined by biochemical fingerprinting, was higher in infants born at home than in those born in hospital (p < 0.01). Several MACs were related to specific lifestyle features, and infants with an anthroposophic lifestyle had a higher proportion of acetic acid and a lower proportion of propionic acid in their stool as compared to the control children. In conclusion, lifestyle factors related to the anthroposophic way of life influenced the composition of the gut flora in the infants. These differences may contribute to the lower prevalence of atopic disease previously observed in children in anthroposophic families.