Jan Källman

Impaired phagocytosis and opsonisation towards group B streptococci in preterm neonates

AIMS To study the chemiluminescence response in polymorphonuclear leucocytes (PMNL) at different stages of maturity and the opsonic capacity of sera with defined titres of anti-capsular type III antibodies, after exposure to serotype III group B streptococci (GBS). The influence of GBS type III capsule expression on PMNL chemiluminescence response was also investigated. METHODS Two clinical isolates of serotype III GBS and two serotype III reference strains which form isogenic variants with high and low amounts of capsule substance, respectively, were used. PMNL and sera were obtained from adult healthy blood donors, full term neonates, and preterm neonates. RESULTS PMNL from premature infants showed a significantly lower chemiluminescence response (p<0.0001) than the PMNL from adults and neonates, while the chemiluminescence response with adult, neonatal, and preterm sera gradually diminished. In the presence of a serum pool with a standardised complement value, raised (>10 mg/l), rather than low (<1.0 mg/l) anti-III antibody titres induced a higher chemiluminescence response to the capsule expressing variant. When GBS were cultured at pH 5.0, the bacteria had a higher buoyant density, reflecting decreased expression of capsule substance compared with bacteria grown at pH 7.4. Concomitantly, there was a substantial increase in chemiluminescence response for all isolates cultured at the lower pH, except for the capsule deficient mutant. CONCLUSIONS PMNL function and opsonic capacity are significantly impaired in neonates and correlate with maturation of the newborn child. The combined defect in cellular and humoral defences in preterm neonates may contribute to their increased susceptibility to GBS infection. Growth conditions for GBS, simulating different in vivo environments, greatly affect capsule expression and resistance to phagocytosis.

Incidence, microbiological findings and clinical presentation of sternal wound infections after cardiac surgery with and without local gentamicin prophylaxis

Sternal wound infection (SWI) is a serious complication after cardiac surgery. In a previous randomized controlled trial, the addition of local collagen-gentamicin in the sternal wound before wound closure was found to significantly reduce the incidence of postoperative wound infections compared with the routine intravenous prophylaxis of isoxazolyl-penicillin only. The aims of the present study were to analyse the microbiological findings of the SWIs from the previous trial as well as to correlate these findings with the clinical presentation of SWI. Differences in clinical presentation of SWIs, depending on the causative agent, could be identified. Most infections had a late, insidious onset, and the majority of these were caused by staphylococci, predominantly coagulase-negative staphylococci. The clinically most fulminant infections were caused by gram-negative bacteria and presented early after surgery. Local administration of gentamicin reduced the incidence of SWIs caused by all major, clinically important bacterial species. Propionibacterium acnes was identified as a possible cause of SWI and may be linked to instability in the sternal fixation. There was no indication of an increase in the occurrence of gentamicin-resistant bacterial isolates in the treatment group. Furthermore, the addition of local collagen-gentamicin reduced the incidence of SWIs caused by methicillin-resistant coagulase-negative staphylococci. This technique warrants further evaluation as an alternative to prophylactic vancomycin in settings with a high prevalence of methicillin-resistant Staphylococcus aureus.

Collagen-gentamicin implant for prevention of sternal wound infection; long-term follow-up of effectiveness

In a previous randomized controlled trial (LOGIP trial) the addition of local collagen-gentamicin reduced the incidence of postoperative sternal wound infections (SWI) compared with intravenous prophylaxis only. Consequently, the technique with local gentamicin was introduced in clinical routine at the two participating centers. The aim of the present study was to re-evaluate the technique regarding the prophylactic effect against SWI and to detect potential shifts in causative microbiological agents over time. All patients in this prospective two-center study received prophylaxis with application of two collagen-gentamicin sponges between the sternal halves in addition to routine intravenous antibiotics. All patients were followed for 60 days postoperatively. From January 2007 to May 2008, 1359 patients were included. The 60-day incidences of any SWI was 3.7% and of deep SWI 1.5% (1.0% mediastinitis). Both superficial and deep SWI were significantly reduced compared with the previous control group (OR=0.34 for deep SWI, P<0.001). There was no increase in the absolute incidence of aminoglycoside resistant agents. The majority of SWI were caused by coagulase-negative staphylococci (CoNS). The incidence of deep SWI caused by Staphylococcus aureus was 0.07%. The results indicate a maintained effect of the prophylaxis over time without absolute increase in aminoglycoside resistance. (ClinicalTrials.gov NCT00484055).

Secretion of IL-6, IL-8 and G-CSF by human endothelial cells in vitro in response to Staphylococcus aureus and staphylococcal exotoxins

The capacity of endothelial cells to produce and release cytokines (IL‐6, IL‐8 and G‐CSF) in response to exposure to Staphylococcus aureus strains or staphylococcal exotoxins (α‐toxin, enterotoxin A and TSST‐1) was investigated. An endothelial cell culture model of human umbilical vein endothelial cells (HUVEC) was used. Five out of ten clinical isolates of S. aureus were found to induce cytokine production and release from endothelial cells. Four of the five isolates that induce cytokine release produced enterotoxin A, B, C, D and/or TSST‐1, compared with two of those that did not induce release. Purified staphylococcal exotoxins (1 pg/ml – 1 μg/ml) did not act as primary stimuli and induced no detectable cytokine secretion. When endothelial cells were prestimulated with IL‐1β or TNFα at a concentration of 1 ng/ml for 2 h, IL‐1β served as a potent primary stimulus for IL‐6, IL‐8 and G‐CSF production, whereas TNFα did not induce any significant cytokine release during the subsequent 24 h. A further increase in IL‐6 and G‐CSF release, but not of IL‐8, was observed when IL‐1β prestimulated cells were exposed to α‐toxin or TSST‐1. However, to potentiate cytokine production (IL‐6 and IL‐8) by SEA, both IL‐1β and the toxin had to be present simultaneously. Our data show that S. aureus, but not staphylococcal exotoxins, have the capacity to act as primary stimuli of endothelial cells and induce production and release of cytokines. IL‐1β may prime HUVEC to release IL‐6, IL‐8 and G‐CSF prior to subsequent stimulation with staphylococcal exotoxins.

Antibiotic Concentrations in Serum and Wound Fluid after Local Gentamicin or Intravenous Dicloxacillin Prophylaxis in Cardiac Surgery

One important aim of antibiotic prophylaxis in cardiac surgery is preventing mediastinitis and thus it would appear to be relevant to study the antibiotic concentrations in pericardial/mediastinal fluid. Local administration of gentamicin in the wound before sternal closure is a novel way of antibiotic prophylaxis and could be effective against bacteria resistant to intravenous antibiotics. This study measured dicloxacillin concentrations in 101 patients in serum and wound fluid following intravenous administration of dicloxacillin. Similarly, concentrations of gentamicin in serum and wound fluid were determined in 30 patients after administration of 260 mg gentamicin in the wound at sternal closure. Median dicloxacillin concentrations in serum and wound fluid at sternal closure were 59.4 and 55.35 mg/l, respectively. Gentamicin levels in the wound were very high (median 304 mg/l), whereas serum concentrations were low (peak median 2.05 mg/l). Dicloxacillin, 1 g given intravenously, according to the clinical protocol, resulted in levels in serum and wound fluid at sternal closure likely to prevent Staphylococcus aureus infections. Locally administered gentamicin resulted in high local concentrations, potentially effective against agents normally considered resistant.

Cost effectiveness of local collagen-gentamicin as prophylaxis for sternal wound infections in different risk groups

Objectives. In a randomized trial addition of local collagen-gentamicin in the sternal wound reduced the rate of sternal wound infection (SWI) to about 50% compared to intravenous prophylaxis alone. The aim of the present study was to evaluate the economic rationale for its use in every-day clinical practice. This includes the question whether high-risk groups that may have particular benefit should be identified. Design. For each patient with SWI in the trial the costs attributable to the SWI were calculated. Risk factors for SWI were identified and any heterogeneity of the effect of the prophylaxis examined. Results. The mean cost of a SWI was about 14500 Euros. A cost effectiveness analysis showed that the prophylaxis was cost saving. The positive net balance was even higher in risk groups. Assignment to the control group, overweight, diabetes, younger age, mammarian artery use, left ventricular ejection fraction < 35% and longer operation time were independent risk factors for infection. Conclusion. The addition of local collagen-gentamicin to intravenous antibiotic prophylaxis was dominant, i.e. resulted in both lower costs and fewer wound infections.

Preliminary results in quantitation of HLA-DRA by real-time PCR: a promising approach to identify immunosuppression in sepsis

IntroductionReduced monocyte human leukocyte antigen (mHLA)-DR surface expression in the late phase of sepsis is postulated as a general biomarker of sepsis-induced immunosuppression and an independent predictor of nosocomial infections.However, traditional monitoring of mHLA-DR by flow cytometry has disadvantages due to specific laboratory requirements. An mRNA-based HLA-DR monitoring by polymerase chain reaction (PCR) would improve the clinical usage and facilitate conduction of large multicenter studies. In this study, we evaluated an mRNA-based HLA-DR monitoring by quantitative real-time PCR (qRT-PCR) as an alternative method to traditional flow cytometry.MethodsFifty-nine patients with sepsis and blood culture growing pathogenic bacteria were studied. Blood samples were collected at day 1 or 2 after admission, for measurement of mHLA-DR by flow cytometry and mRNA expression of HLA-DRA and class II transactivator (CIITA) by qRT-PCR. Blood samples from blood donors were used as controls (n = 30).ResultsA significant reduced expression of mHLA-DR, HLA-DRA, and CIITA was seen in septic patients compared with controls. HLA-DRA mRNA level in whole blood was highly correlated with surface expression of mHLA-DR.ConclusionsPatients with sepsis display a diminished expression of HLA-DR at the monocyte surface as well as in the gene expression at the mRNA level. The mRNA expression level of HLA-DRA monitored by qRT-PCR correlates highly with surface expression of HLA-DR and appears to be a possible future biomarker for evaluation of immunosuppression in sepsis.

Adherence of group B streptococci to human endothelial cells in vitro.

A model using human umbilical vein endothelial cells was developed to study the adhesion of group B streptococci. Ten clinical isolates of serotypes Ia, Ib and III, including six blood isolates from neonates with early‐onset disease, were studied. Isogenic variants with different ability to express capsule substance were also included. Clinical isolates of serotype III adhered significantly better than other serotypes. Strains with high ability to adhere to endothelial cells also had high ability to bind to plastic. Isogenic variants of serotype III with low amounts of capsule substance adhered significantly better to cells than variants expressing high amounts of capsule substance. These results show that the higher adherence of serotype III strains may be a virulence factor and contribute to the finding that this serotype dominates in invasive group B streptococcal disease.

Penetration of Group B Streptococci through Polarized Madin-Darby Canine Kidney Cells

Group B streptococci (GBS) are one of the major causes of invasive neonatal infection. The pathogenesis of early onset disease is a multistep process. Adhesion of GBS to eucaryotic cells is considered to be an important step for the establishment of infection. Subsequent to adhesion, GBS invade cells and give rise to septicemia and meningitis. To investigate passage of GBS across epithelial cell linings we examined the interaction between bacteria and Madin-Darby canine kidney (MDCK) cells. When grown on permeable support, these cells form a polarized epithelial monolayer with an apical-to-basolateral orientation, which more reflects the in vivo situation compared with conventionally cultured cells. Our results show that GBS are translocated in vacuoles from the apical to the basolateral surface of MDCK cells in a temperature-dependent process. The passage of GBS through the cells is selective with only small numbers of bacteria penetrating in the basolateral-to-apical direction. Transcytosis of GBS starts before decrease in transepithelial resistance of the monolayer. These data suggest a mechanism for traversal of GBS over intact chorioamniotic membranes and from alveoli into the circulation of the fetus.

Coagulase‐negative staphylococci isolated from sternal wound infections after cardiac surgery: attachment to and accumulation on sternal fixation stainless steel wires

Sternal wound infection (SWI) is a serious complication after cardiac surgery. Coagulase‐negative staphylococci (CoNS) have been found to be the most common pathogen involved in this postoperative infection related to implanted foreign materials, i.e. sternal fixation wires made from stainless steel. In this study a rapid and simple assay was developed for studying attachment and accumulation of CoNS on stainless steel wires in vitro using [3H] thymidine. The method showed a potential to detect differences in the dynamics of the adherence patterns among various CoNS isolates. However, no differences in attachment and accumulation were found between isolates causing deep SWI after cardiac surgery and contaminant isolates. In addition, there were no differences in the distribution of the ica operon between the two groups, as determined by polymerase chain reaction (PCR). Nevertheless, the ability to produce biofilm was found to be present significantly more frequently among SWI isolates than among contaminants.