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.

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.

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).

Exogenous or endogenous reservoirs of nosocomial Pseudomonas aeruginosa and Staphylococcus aureus infections in a surgical intensive care unit.

OBJECTIVE: A 4 month prospective study was performed to assess the incidence and routes of endogenous or exogenous colonization and nosocomial infection caused by Staphylococcus aureus and Pseudomonas aeruginosa in surgical critically ill patients. DESIGN: A total of 4634 specimens were obtained. Patient's nasal, scalp, and rectal swabs as well as tracheal secretion (TS) were cultured every second day beginning on the day of admission. Nasal swabs and hand cultures of the personnel as well as cultures from gowns were also taken. All isolates of S. aureus were phage typed and 116 of these isolates were also plasmid typed. P. aeruginosa isolates were sero- and pyocin typed. Resistance patterns were determined in all isolates. SETTING: The study was carried out in the surgical intensive care unit (SICU) of an teaching hospital. PATIENTS: During the study period each patient (a total of 153 patients) admitted to the SICU entered the study. RESULTS: P. aeruginosa and S. aureus colonisation rate on admission were 5% and 36.5% respectively. Only 10 patients (6.5%) were colonized with P. aeruginosa during hospitalization, and only 7 patients (4.5%) acquired S. aureus in the surgical intensive care unit (SICU). The most common primary colonisation site of P. aeruginosa was the rectum, whereas S. aureus was predominantly found in nasal cultures. Horizontal transmission of S. aureus occurred in only 2 patients. CONCLUSION: The study suggests that colonisation with P. aeruginosa and S. aureus occurs from endogenous rather than from exogenous sources and that the endogenous acquisition of both bacteria play a more important role in development of nosocomial infections than the exogenous route of transmission.

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.

Comparison of three typing methods in hospital outbreaks of Acinetobacter calcoaceticus infection.

During a period of 11 months Acinetobacter baumanii was isolated from 27 patients and 21 environmental samples in the Intensive Care Unit (ICU) and in one surgical unit of a University Hospital. The isolates were characterized by biotyping, antibiograms and plasmid profiles and compared with co-isolates. Plasmid fingerprinting distinguished three outbreaks, whereas other typing methods were less sensitive and discriminatory. Although plasmid profiles seem to be a simple and reproducible marker for epidemiological studies with acinetobacter strains, it might be useful to combine at least two typing methods since plasmids are unstable genetic structures, and not all strains possess plasmids.

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.

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 activity of fleroxacin and 14 other antimicrobials against slime- and non-slime-producing Staphylococcus epidermidis.

The in vitro activity of the 4-quinolone compound fleroxacin (Ro-23-6240) was compared with that of 14 other antimicrobials against a total of 50 recent clinical isolates of 25 slime- and 25 non-slime-producing coagulase-negative staphylococci. Susceptibility testing (MIC/MBC) was performed by a microtiter broth dilution technique and the combination effect of fleroxacin plus rifampin was studied by checkerboard titration in microtiter trays. Fleroxacin inhibited the most slime- and non-slime producing coagulase-negative staphylococci at MIC90 0.25 and I micrograms/ml, respectively. Overall fleroxacin was as active or even better as ofloxacin, cefotiam, cefazolin, cefamandole, clindamycin or vancomycin but 2- to 8-fold less active than rifampin. The fleroxacin-rifampin combination was indifferent in 17%, additive in 78.7% and synergistic in 4.3%.

In vitro activity of fleroxacin and 6 other antimicrobials against Acinetobacter anitratus.

The in vitro activity of the 4-quinolone compound fleroxacin (Ro-23-6240) was compared with that of enoxacin, ofloxacin, cefepime (BMY-28142), ceftazidime, ceftriaxone, and tobramycin against a total of 30 recent clinical isolates of Acinetobacter calcoaceticus subsp. anitratum. Susceptibility testing (MIC50/MIC90) was performed by a microtiter broth dilution method and the combination effect of ceftriaxone plus tobramycin was studied by checkerboard titration in microtiter trays. Fleroxacin inhibited most A. calcoaceticus subsp. anitratum at 1 microgram/ml and was as active as enoxacin or tobramycin but slightly less active than ofloxacin (MIC50 = 0.25 microgram/ml; MIC90 = 2.5 microgram/ml) or cefepime (BMY-28142: MIC50 = 0.25 microgram/ml; MIC90 = 1 microgram/ml). Ceftazidime and ceftriaxone were inactive (MIC90 = 8 micrograms/ml and 32 micrograms/ml, respectively). The combination of ceftriaxone plus tobramycin was synergistic in 16.7%, additive in 60%, and indifferent in 23.3%.

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.

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.

Penetration into tissues of various drugs active against gram-positive bacteria.

Gram-positive bacteria are the most important pathogens causing hospital- and community-acquired infections. We therefore reviewed the penetration of various antibiotics active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus into tissues, where staphylococcal infections are common. Rifampicin reaches heart valve concentrations of 65% of the simultaneous serum levels. At 8 h after administration blood and tissues concentrations of rifampicin exceeded the MIC90 values for S. aureus as well as for S. epidermidis. After a 2-g intravenous bolus injection of flucloxacillin heart valve concentrations exceeded MIC values for staphylococci for more than 8 h whereas subcutaneous and muscle concentrations declined within the same time to undetectable levels. The MIC90 values of vancomycin for S. epidermidis and Enterococcus faecalis are 2.0 and 4.0 mg/l respectively and for S. aureus 1.0 mg/l. This concentration is reached in subcutaneous tissue, heart valves and muscle for at least 4-6 h after administration of 15 mg/kg, however the corresponding value for Enterococcus faecalis in heart valve is maintained only for 3-4 h. After two and three dose regimens of teicoplanin serum and bone levels were significantly higher than fat levels, exceeding the MIC90 values for S. aureus, S. epidermidis and E. faecalis. The ratio of tissue concentration of teicoplanin to serum concentrations was 11% for fat and 65% for bone.

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.

Combination effect of SCE-2787 and cefepime with aminoglycosides on nosocomial gram-negative bacteria.

The in vitro activity of cefepime and SCE-2787, two new parenteral cephalosporins, and the combination effect with tobramycin and gentamicin against nosocomial gram-negative rods was studied using checkerboard agar dilution technique. Cefepime showed excellent in-vitro activity against Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens and Proteus vulgaris (MIC90 0.03-0.125 mg/l) and good to moderate activity against Acinetobacter anitratus, Pseudomonas aeruginosa and Pseudomonas cepacia (MIC90 4-16 mg/l). SCE-2787 had an excellent activity against Citrobacter spp. (MIC90 0.125 mg/l) and a very good activity against A. anitratus, P. aeruginosa and P. vulgaris (MIC90 1-2 mg/l). Pseudomonas maltophilia was not inhibited at therapeutically achievable concentrations (MIC90 64 mg/l). On average, 14-28% of the strains were inhibited by synergistic SCE-2787 aminoglycoside-combinations, whereas only 8.6% were inhibited by a synergistic effect of the combination with cefepime and gentamicin. No antagonism occurred with any of the combinations.

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.

In vitro susceptibility of methicillin-resistant Staphylococcus aureus and slime-producing and non-slime-producing coagulase-negative staphylococci to fusidic acid.

The in vitro susceptibility of 100 oxacillin-resistant Staphylococcus aureus and 100 oxacillin-resistant coagulase-negative staphylococci (CNS; 50 slime-negative and 50 slime-positive strains) was determined by agar dilution technique, with and without the addition of 50% human serum. All strains tested were highly sensitive to fusidic acid. S. aureus and CNS showed MIC50 values of 0.125 and 0.25 mg/l, respectively. MICs of all strains increased significantly in the presence of 50% human serum. Only minor differences were noted between the MICs of slime-producing and slime-deficient CNS.