In vitro and in vivo activity of hyperimmune globulin preparations against multiresistant nosocomial pathogens.

PURPOSE: We compared different immunoglobulin preparations containing IgG (Intraglobin/Intratect) or a mixture of IgG, IgA, and IgM (Pentaglobin) to assess the opsonic and protective efficacy of human immunoglobulin preparations against multiresistent nosocomial pathogens. MATERIALS AND METHODS: Clinical isolates of E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus were tested by opsonophagocytic assay using immunologobulin preparations at dilutions usually obtained in patients. The target antigens of opsonic antibodies were characterized by opsonophagocytic inhibition assays, and the protective efficacy in vivo was tested in a mouse bacteremia model as previously described. RESULTS: All strains were killed to at least 50% by Pentaglobin. One P. aeruginosa strain was not efficiently killed by Intraglobin (23%) but the other strains were killed by Intraglobin to a similar degree compared to Pentaglobin. Opsonic IgG antibodies against E. faecalis were directed against LTA, while opsonic antibodies in Pentaglobin were primarily directed against other cell wall carbohydrates. In a mouse bacteremia model, Pentaglobin was more protective than Intratect against Staphylococcus aureus, while Intratect reduced colony counts better than normal rabbit serum or saline. CONCLUSIONS: All tested human immunoglobulin preparations contain opsonic and protective antibodies against targets present on multiresistant Gram-positive and Gram-negative bacteria. Enrichment of these preparations with IgM increases the protective efficacy against some strains, probably due to antibodies directed against cell wall carbohydrates.

Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations.

Three different commercially available polyvalent immune globulins (IG) were investigated for the existence of antibodies against cell wall carbohydrates of four different E. faecalis serotypes (using a cell wall carbohydrate-enzyme-linked immunosorbent assay), and whether these antibodies mediated opsonic killing (using an opsonic-killing assay). All three IG preparations contained antibodies against all four serotypes (CPS-A to CPS-D). However, only one of the three IG preparations showed opsonic killing against all four serotypes. Average killing was higher against serotypes A and B (72 and 79 %, respectively) than against serotypes C and D (30 and 37 %, respectively). Such IG preparations could play a role as an adjuvant therapeutic option in life-threatening infections with E. faecalis, particularly when resistant strains are involved.

Light fluorous synthesis of glucosylated glycerol teichoic acids.

We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a terminal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers featuring a terminal alcohol functionality. These complementary linker systems have been developed because the nature of the TA chain terminus can play a role in the biological activity of the synthetic TAs. A novel α-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust light fluorous synthetic protocol.

Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections.

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Enterococci, especially Enterococcus faecium, are well-known pathogens of hospitalized patients and are frequently linked with resistance against multiple antibiotics, which compromises effective therapy. Rabbit immune serum raised against heat-killed E. faecium E155, a HiRECC clone, was used in an opsonophagocytic assay, an inhibition assay and a mouse bacteraemia model to identify targets of opsonic and protective antibodies. Serum against whole heat-killed bacteria was opsonic and recognized a protein of about 72 kDa that was abundantly secreted. This protein, identified as SagA by LC-ES-MS/MS, was expressed in Escherichia coli and purified. Rabbit serum raised against the purified protein showed opsonic killing activity that was inhibited by almost 100% using 100 µg purified protein ml(-1). In a mouse bacteraemia model, a statistically significant reduction of the colony counts in blood was shown with immune rabbit serum compared with preimmune serum using the homologous and a heterologous vancomycin-resistant enterococci (VRE) strain. These results indicate that SagA could be used as a promising vaccine target to treat and/or prevent VRE bacteraemia.

Enterococcal surface protein contributes to persistence in the host but is not a target of opsonic and protective antibodies in Enterococcus faecium infection.

Enterococci are important nosocomial pathogens with multiple intrinsic and acquired resistances to antibiotics. In the past, the majority of infections were caused by Enterococcus faecalis; however, an increase in Enterococcus faecium clinical isolates has been observed in recent years. The enterococcal surface protein (Esp) is expressed on the surface of most E. faecium clinical isolates and has been shown to be involved in biofilm formation. Here, E. faecium E1162 and its previously created insertion-deletion mutant of the esp gene, E. faecium E1162Deltaesp, were compared in a mouse bacteraemia model. Anti-Esp serum was tested for its capacity to mediate opsonophagocytic killing of E1162 in vitro and to protect against E. faecium bacteraemia. The inactivation of esp attenuated E. faecium virulence with reduced numbers of bacteria recovered from the kidneys in animals infected with the mutant compared to the wild-type strain (P=0.035). Passive immunization with rabbit polyclonal serum raised against the recombinant N-terminal Esp protein did not protect mice against E. faecium bacteraemia (P>0.05). In contrast, mice passively immunized with polyclonal antiserum raised against lipoteichoic acid (LTA) from E. faecalis had lower numbers of E. faecium E1162 in the blood compared to mice immunized with normal rabbit serum. These results suggest that Esp contributes to E. faecium persistence in the host. However, in contrast to LTA, Esp does not seem to be a target for protective antibodies in E. faecium strain E1162 in mouse bacteraemia.

In vitro assessment of the host response against Enterococcus faecalis used in probiotic preparations.

Along with other lactic acid bacteria, enterococci are used in food products and as health promoting agents. The safety of these products must be ensured, because they contain potentially pathogenic microorganisms. Here we present an in vitro opsonophagocytic assay that closely mimics the protective human immune response to Enterococcus faecalis and Enterococcus faecium. A collection of closely related E. faecalis isolates used as probiotics showed different susceptibilities to opsonic killing, suggesting that some of these isolates possess a capsule while other do not. This information may be helpful in assessing the safety of a given bacterial isolate used and could detect likely enterococcal candidates for probiotic preparations.

In vitro susceptibility to everninomycin of gram-positive nosocomial pathogens isolated from intensive care units in Germany.

BACKGROUND: Gram-positive bacteria are increasingly found to be causative pathogens in nosocomial infections, and the occurrence of vancomycin resistance in enterococci as well as staphylococci has prompted the investigation of alternative antimicrobial agents active against these strains. Everninomycin, a new oligosaccharide antibiotic, has excellent in vitro activity against gram-positive bacteria, including those resistant to vancomycin. However, avilamycin, a related compound, has been used in Europe as a growth promoter in animal food for years and concern has been raised that cross-resistance in clinical isolates may arise. METHODS: We studied a collection of 268 nosocomial gram-positive isolates from intensive care unit patients with nosocomial pneumonia, urinary tract infection and sepsis, using standard in vitro susceptibility testing. RESULTS: It could be shown that all species tested were exquisitely sensitive to everninomycin (MIC(90) of 0.38 microg/ml for Staphylococcus aureus, 0.5 microg/ml for enterococci and 0.75 microg/ml for coagulase-negative staphylococci). Furthermore, no difference could be observed between methicillin-resistant and methicillin-sensitive S. aureus or between Enterococcus faecium and Enterococcus faecalis. CONCLUSIONS: These results suggest that everninomycin is a promising antibiotic for the treatment of nosocomial infections in intensive care unit patients and that the use of a related substance as an additive in animal food has not yet promoted resistance in clinical isolates.

Estimation of extra hospital stay attributable to nosocomial infections: heterogeneity and timing of events.

Infections acquired in hospital are likely to affect the duration of hospitalization. Suitable statistical methods for estimating the extra days spent in hospital due to nosocomial infections should allow modeling of the heterogeneity of the patient population and the timing of events, as failure to account for important covariates and failure to model adequately the timing of events may lead to biased results. Three approaches have been used in the past to estimate the extra stay: a comparison of duration of stay of infected and uninfected patients, matching of infected and uninfected patients with respect to potentially important determinants of the length of hospital stay, and matching for time-to-infection in addition to the other factors. While these approaches can allow for the heterogeneity of the patient population, none takes sufficient account of the real timing of events and may overestimate the effect of nosocomial infections. We explored the statistical methods available for analyzing time-to-event data and derived alternative methods to estimate the extra stay that appropriately account for heterogeneity and timing. Data from two prospective cohort studies on postoperative wound infection and on nosocomial pneumonia showed that the two-group comparison yields the highest estimates of extra stay (21 and 14 extra days), while matching for confounders and time reduced the estimates to 11 and 8 extra days; our methods yield even lower results (10-12 and 3-4 extra days).

Differential cytokine production in stimulated blood cultures from intensive care patients with bacterial infections.

Mice infected with bacteria develop an interferon-gamma (IFN-gamma) dependent hypersensitivity to lipopolysaccharide (LPS) and other bacterial components. The broader aim of this study is to find out whether such hypersensitivity also occurs in patients suffering from bacterial infections. The capacity of stimulated peripheral blood cells from infected, intensive-care patients to produce cytokines (IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)) was compared to that of healthy donors. Culturing of the cells was carried out preferentially in whole blood diluted 1:3. Whole blood cultures (WBC) were stimulated with lipopolysaccharide (LPS), whole killed Salmonella typhimurium and Staphylococcus aureus and concanavalin A (ConA), and the cytokine production was determined. Two main findings emerged from this study: The IFN-gamma production by WBC of patients was, compared to healthy donors, markedly suppressed, regardless of stimulus used. Further, patients' WBC exhibited a suppressed TNF-alpha production after stimulation with LPS. Surprisingly, following stimulation with bacteria (S. typhimurium and S. aureus) an elevated TNF-alpha and IL-6 response was obtained. Thus, in severely infected patients the cytokine responses of peripheral blood cells to LPS may be suppressed, while the response to other bacterial components is enhanced.

Scoring system for nosocomial pneumonia in ICUs.

OBJECTIVE: To develop a scoring system for stratifying patients in intensive care units (ICUs) by risk of developing nosocomial pneumonia (NP), based on variables generally available in an ICU, and to determine the probability of a patient developing NO in the ICU. DESIGN AND SETTING: a 2-year prospective cohort study conducted in a medical and surgical ICU. PATIENTS: 756 patients admitted to the ICU for 48 h or more were followed up until the development of NP or death or discharge from the ICU. MEASUREMENTS AND RESULTS: 129 (17.1%) patients developed NP, 106 (14%) in the first 2 weeks. The following independent risk factors were identified by multivariate analysis: no infection on admission [relative risk (RR) = 3.1, 95% confidence intervals (CI) = 2.0 to 4.81; thorax drainage (RR = 2.1, 95% CI = 1.2 to 3.5); administration of antacids (RR = 2.1, 95% CI = 1.4 to 3.1); partial pressure of oxygen (PO2) > 110 mmHg (RR = 1.6, 95% CI = 1.0 to 2.6); administration of coagulation factors (RR = 1.8 95% CI = 1.0 to 3.2); male gender (RR = 2.7, 95% CI = 1.2 to 6.3); urgent surgery (RR = 2.4, 95% CI = 0.9 to 6.4); and neurological diseases (RR = 4.2, 95% CI = 1.9 to 9.4). To obtain a predictive risk index for NP, a scoring system was developed using a multivariate model. The probability of developing NP varied between 11.0% in the lowest risk group and 42.3% in the highest risk group. The patients' risk of acquiring NP was seven times higher in the highest score category (i.v.) than in the lowest one (I). CONCLUSIONS: ICU patients can be stratified into high- and low-risk groups for NP. No infection on admission, thorax drainage, administration of antacids, and PO2 > 110 mmHg were associated with a higher risk of NP during the entire 2-week period.

Cross infections due to coagulase-negative staphylococci in high-risk patients.

Until recently, infections due to coagulase-negative staphylococci (CNS) have been regarded as endogenous in origin. However, there are now increasingly reports in the literature on the endemic occurrence of distinct strains of CNS. Several outbreaks due to CNS are reported in cardiac surgery or in neonates. The latter seem to be high risk populations in regard to CNS infections because of certain risk factors (i.e. degree of immunosupression, routine use of central venous catheters and parenteral lipids as well as broad spectrum antibiotic therapy). On the other hand, these newborn babies have no physiological skin flora and are therefore easily colonized by multiresistent bacteria. The persistence of certain well-defined Staphylococcus epidermidis (SE) strains in neonatal intensive care units have been demonstrated over periods as long as a decade. Specific putative virulence factors (i.e. slime production and polysaccharide/adhesin PS/A) were more common in endemic strains as compared to single isolates. Pulsed-field gel electrophoresis (PFGE) proves to be a powerful tool in the study of the epidemiology of CNS while other modern typing techniques (ribotyping, plasmid typing) were also used in the literature to investigate outbreaks of CNS infections.

Synergy of simultaneous administration of ofloxacin and granulocyte colony-stimulating factor in killing of Escherichia coli by human neutrophils.

The in vitro effect of subinhibitory and inhibitory concentrations of ofloxacin and G-CSF on the bactericidal activity of polymorphonuclear leucocytes (PMNL) against Escherichia coli was investigated. PMNL obtained from healthy volunteers were incubated with different concentrations of G-CSF and ofloxacin for 180 min. The minimum inhibitory concentration (MIC) of ofloxacin and even 1/4 x MIC enhanced the bactericidal activity of PMNL. G-CSF at a concentration of 6,000 units/ml led to a significant improvement of the bactericidal activity of PMNL. The combination of 6,000 units/ml of G-CSF and ofloxacin in inhibitory as well as subinhibitory concentrations, however, showed a significant synergistic effect on the antibacterial activity of PMNL during the complete incubation period. Combinations of G-CSF and antibiotics could therefore be beneficial for infected patients, especially those with impaired cellular host defense.

Glycopeptides in the treatment of staphylococcal infections.

Gram-positive bacteria are rapidly becoming the most important pathogens in nosocomial infections. In recent years, attention and concern have been focused on the gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. These microorganisms are well equipped to exert their pathogenic effects and to display virulence. Treatment of severe infections caused by gram-positive bacteria remains difficult because of the increase in infections caused by methicillin-resistant staphylococci, and this has renewed interest in the glycopeptide antibiotics, vancomycin and teicoplanin. According to National Nosocomial Infection Surveillance Study data, in 1989, 60% of coagulase-negative staphylococci and 22% of Staphylococcus aureus strains showed methicillin resistance. Among other factors, successful antimicrobial therapy depends on rapid and reliable antibiotic delivery to the infection site at a concentration adequate to inhibit the majority of infecting organisms. Glycopeptides may be important in the therapy of catheter-related infections, which are mainly caused by coagulase-negative staphylococci and Staphylococcus aureus.

Combination effect of meropenem with aminoglycosides and teicoplanin on Pseudomonas and enterococci.

The in vitro activity of meropenem, a new carbapenem, and the combination effect with netilmicin, tobramycin, gentamicin, and teicoplanin against Pseudomonas spp. and enterococci was studied. Meropenem showed very good in vitro activity against Pseudomonas aeruginosa (MIC90 2 mg/l) and good to moderate activity against Pseudomonas putida (MIC90 4 mg/l) and Enterococcus faecalis (MIC90 8 mg/l). Aminoglycosides were highly active against P. putida (MIC90 0.5 mg/l), but showed only moderate activity against P. aeruginosa. The synergistic effect of meropenem was shown in combination with teicoplanin against E. faecalis (40%). No Pseudomonas strains were inhibited by the synergistic effect of meropenem with aminoglycosides. No antagonism occurred with any of the combinations.

Endemic nosocomial transmission of Staphylococcus epidermidis bacteremia isolates in a neonatal intensive care unit over 10 years.

To assess long-term nosocomial transmission, trends in antibiotic resistance, and expression of potential virulence factors, 86 randomly selected Staphylococcus epidermidis bloodstream isolates obtained from 80 patients in a neonatal intensive care unit (NICU) over a 10-year period were studied. Pulsed-field gel electrophoresis (PFGE) analysis of SmaI-digested whole chromosomal DNA revealed distinctive banding patterns that persisted in the NICU over long periods. Pattern A included 22 isolates (26%) obtained during 1983-1990, and pattern B included 24 isolates (28%) from 1983 to 1991. All 10 isolates examined in 1984 fell into one of these two patterns. Isolates with either pattern expressed polysaccharide/adhesin (PSA) and slime; 90% and 87% were resistant to oxacillin and gentamicin, respectively, with no trends over time. These findings suggest that distinct clones of S. epidermidis can become endemic in NICUs over periods as long as a decade and that nosocomial transmission plays an important role in neonatal S. epidermidis bacteremia.

Serum bactericidal activity after administration of four cephalosporins in healthy volunteers.

Serum bactericidal activity (SBA) was determined against ten strains each of Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris and Enterobacter cloacae in six volunteers 1 h and 4 h after intravenous infusion of 1 g and 2 g cefotaxime and cefmenoxime, and 2 g flomoxef, and against ten strains of Pseudomonas aeruginosa after infusion of 1 g and 2 g ceftazidime. Flomoxef showed the highest SBA against methicillin-susceptible Staphylococcus aureus. All cephalosporins had high SBA against gram-negative rods. Cefotaxime had the highest SBA against Klebsiella pneumoniae and Enterobacter cloacae. The SBA against Pseudomonas aeruginosa after 1 g and 2 g doses of ceftazidime was very similar.

MIC and serum bactericidal activity of clindamycin against methicillin-resistant and -sensitive staphylococci.

Six volunteers were given 600 mg clindamycin intravenously to investigate the serum bactericidal activity (SBA) against 50 methicillin susceptible (MSSA) and 50 methicillin resistant Staphylococcus aureus (MRSA) strains. Minimal inhibitory concentrations (MIC) against MSSA, MRSA and 50 methicillin resistant strains of Staphylococcus epidermidis (MRSE), of which 50% were slime-producing, were determined. SBA of clindamycin against MSSA and MRSA was equally high (mean reciprocal SBA titer against MSSA vs MRSA 1 h after application was 13.0 vs 13.45), although MICs against MRSA were markedly higher than against MSSA (MIC 90 of MRSA vs MSSA: 0.06 vs > 32 mg/l). There was no difference in MICs between slime- and non-slime-producing MRSE.

Pseudomonas aeruginosa in a neonatal intensive care unit: reservoirs and ecology of the nosocomial pathogen.

Pseudomonas aeruginosa is one of the leading nosocomial pathogens. In hospitals, organisms are commonly recovered from moist environments. To determine the reservoir and population dynamics of particular strains, highly discriminative typing methods are required. Hybridization of enzymatically restricted P. aeruginosa DNA with two gene probes led to the identification of infecting and colonizing strains prevalent over a 5-month period in a neonatal intensive care unit. Four genotypically distinct strains were repetitively isolated from tap water from several faucets on the ward. P. aeruginosa isolates recovered from tap water on adjacent wards supplied by the same water system had different genotypes, while samples taken from the mains were negative for the organism. Serotyping of O antigens showed variable reproducibility and could not elucidate the strain-specific reservoirs. It is concluded that organisms are transmitted horizontally between faucets and prevail in reservoirs for prolonged periods.

In vitro activity of sodium bisulfite and heparin against staphylococci: new strategies in the treatment of catheter-related infection.

Device-related infections often persist until the device is removed because systemic antibiotic therapy fails frequently. In an in vitro model, the activity of sodium bisulfite (an antioxidant added to many drugs) heparin with chlorbutol or chlorcresol as preservatives, and their combination with cefpirom for treating the internal surface of polyurethane and silicone catheters colonized with Staphylococcus aureus and Staphylococcus epidermidis was studied. Sodium bisulfite (0.05%) markedly reduced catheter colonization. Heparin combined with chlorbutol, 1000 micrograms/mL of cefpirom, and 0.05% of sodium bisulfite was not more effective than sodium bisulfite alone. A lesser effect was shown by treating adherent bacteria on both catheters with heparin plus preservatives alone. Not only antibiotics but also other substances with antibacterial activity should be evaluated for treatment and prophylaxis of catheter-related infection.

In vitro activity of vancomycin and teicoplanin against Staphylococcus aureus and Staphylococcus epidermidis colonizing catheters.

In a quantitative in vitro model the activity of vancomycin and teicoplanin in two concentrations (4 x MBC and 1 mg/l) against Staphylococcus aureus and a slime-producing Staphylococcus epidermidis strain colonizing the internal surface of polyurethane and silicone catheters was studied. In comparison with vancomycin, teicoplanin achieved a significantly greater reduction (p < 0.05) in the counts of Staphylococcus aureus and Staphylococcus epidermidis adhering to both polyurethane and silicone catheters.