Erik Kihlström

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.

A clinical and epidemiological study of "ornithosis" caused by Chlamydia psittaci and Chlamydia pneumoniae (strain TWAR).

Ornithosis is a notifiable disease in Sweden since 1954. In 1981 and 1982 a sharp increase in the number of notifications occurred. Since then the number has declined but is still high. A changed epidemiology characterized by no history of bird contact and no common source, raised the suspicion of a new agent. Serological data now suggest that the epidemic was to a substantial part due to Chlamydia pneumoniae (strain TWAR) (48% of the patients during 1981-1982 compared to 9% during 1984-1987). During recent years TWAR infections have thus become uncommon but reappearance can be expected in the near future. The clinical picture as well as the complications appear to be very similar in infections caused by C. pneumoniae and C. psittaci.

PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid

PLUNC (palate, lung and nasal epithelial clone) is a newly discovered gene that is expressed in the upper respiratory tract and is suggested to be of importance in host defence against bacteria. We have identified two forms of the PLUNC protein in human nasal lavage fluid (NLF) using two-dimensional gel electrophoresis (2-DE) and MS. The apparent molecular masses and isoelectric points of these forms are 24.8 kDa/pI 5.4 and 25.1 kDa/pI 5.5. Notably, the 24.8 kDa/pI 5.4 form of PLUNC is an abundant protein in the 2-DE protein patterns of NLF from healthy subjects. Decreased levels of PLUNC were found in NLF from smokers and workers exposed to reactive epoxy chemicals, indicating that long-term exposure to airway irritants impairs the production of PLUNC in the upper respiratory tract. We have also investigated the presence of lipopolysaccharide (LPS)-binding proteins in NLF. Five proteins were found to adsorb to a LPS-coated surface; two of these proteins correspond to the two PLUNC forms, as judged by 2-DE pattern matching. For comparison, human saliva was found to contain a set of LPS-binding proteins with similar 2-DE spot positions (the same pIs but somewhat lower apparent molecular masses of approximately 20 kDa). These results indicate that PLUNC may be a new marker of airway inflammation and may play a part in the innate immune response, and that human saliva contains yet other members of the family of LPS-binding proteins.

Increase of staphylococci in neonatal septicaemia: a fourteen-year study

All cases of neonatal septicaemia during 1981‐94 were studied at Örebro Medical Centre Hospital, Sweden. One hundred and thirty‐two children fulfilled laboratory and clinical criteria for neonatal septicaemia and were included. Staphylococcus aureus (n= 41), Group B streptococcus (GBS) (n= 32) and coagulase‐negative staphylococci (CoNS) (n= 27) were the dominating aetiologies. The annual incidence of septicaemia increased significantly, from 2.3 cases during the first 7‐year period to 3.3 per 1000 live births during 1988‐94. This increase was caused by S. aureus and CoNS, which mainly affected premature children and had an onset more than 48 h after delivery. GBS, on the other hand, slightly decreased and affected full‐term children within 48 h. The overall mortality was 11%. CoNS isolated during the latter 7‐year period were more resistant to antibiotics than those isolated during 1981‐87; resistance to methicillin increased from 14 to 45% and to gentamicin from 0 to 20%. These changes in aetiology and antibiotic susceptibility should be considered when selecting antibiotic treatment in neonatal septicaemia.

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.

Infection of Human Endothelial Cells with Staphylococcus aureus Induces Transcription of Genes Encoding an Innate Immunity Response

Staphylococcus aureus is a gram‐positive bacterium frequently isolated from patients with bloodstream infections. Endothelial cells (EC) play an important role in host defence against bacteria, and recent reports have shown that infection of EC with S. aureus induces expression of cytokines and cell surface receptors involved in activating the innate immune response. The ability of S. aureus to invade nonphagocytic cells, including EC, has been documented. However, the knowledge of the role of EC in pathogenesis of S. aureus infection is still limited. In this study, we investigate the gene‐expression program in human EC initiated by internalized S. aureus, using microarray analysis. We found 156 genes that were differentially regulated at least threefold, using arrays representing 14,239 genes. Many of the upregulated genes code for proteins involved in innate immunity, such as cytokines, chemokines and cell adhesion proteins. Other upregulated genes encode proteins involved in antigen presentation, cell signalling and metabolism. Furthermore, intracellular bacteria survived for days without inducing EC death.

Yersinia enterocolitica Septicemia: Clinical and Microbiological Aspects

Septicemia is a rare but serious complication of infection with Yersinia enterocolitica (Y.e.). Seven cases of Y.e. septicemia are presented. Five of the patients had no underlying disease predisposing to septicemia. Five patients displayed recurrent episodes of septicemia, despite treatment with recommended doses of antibiotics to which the isolates were sensitive in vitro. One patient developed endocarditis which required surgical replacement of the aortic valve. Other clinical manifestations were arthritis, diverticulitis and pulmonary abscesses. The outcome was fatal to 3 elderly patients. The serological response to Y.e. was followed by tube agglutination and a diffusion-in-gel enzyme-linked immunosorbent assay. One patient, with a benign course of illness, had transient elevated Y.e. antibody titres, while the 3 cases with a protracted disease showed sustained antibody responses for 6-18 months. Blood isolates of Y.e. had ordinary virulence characteristics identical to fecal isolates and produced extracellular beta-lactamase. All isolates were sensitive in vitro to trimethoprim-sulfamethoxazole, mecillinam, piperacillin, cefotaxime, ceftazidime, chloramphenicol and gentamicin. The lowest MIC values were recorded for mecillinam. Full synergistic activity was demonstrated when mecillinam was combined with trimethoprim-sulfamethoxazole, cefuroxime or rifampicin.

Expression of chemokines and adhesion molecules in human coronary artery endothelial cells infected with Chlamydia (Chlamydophila) pneumoniae

Chlamydia pneumoniae has during recent years been associated with cardiovascular disease and atherosclerosis. Chemokines, leukocyte adhesion proteins and metalloproteinases are significant for chemotaxis and attachment of leukocytes to vessel walls, and for stability of atherosclerotic plaques. To determine the ability of C. pneumoniae to elicit inflammation in a relevant target host cell, we infected human coronary artery endothelial cells (HCAEC) with a clinical isolate of C. pneumoniae. Extracellular release of five chemokines, two adhesion proteins and a metalloproteinase was measured at different time points after infection using a cytometric bead assay and ELISA. Secretion of IL‐8, MCP‐1, MIG, IP‐10 and ICAM‐1 was significantly increased 48 h after C. pneumoniae infection of HCAEC in comparison with uninfected controls. Release of RANTES occurred already 6 h after infection. C. pneumoniae did not elicit release of E‐selectin or MMP‐1. We conclude that C. pneumoniae induces expression of proinflammatory components in HCAEC, which would promote migration of leukocytes towards endothelial cells. This suggests that C. pneumoniae initiates and propagates vascular inflammation in ways that contribute to coronary artery disease.

Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes

Broad range PCR amplification and genus‐specific 16S ribosomal DNA hybridization was used to demonstrate that Chlamydia, Helicobacter and Mobiluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR product obtained from pure cultures of the three genera. The sensitivity of the assay was determined by analysis of C. trachomatis serially diluted in urine. The detection limit was 1–10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S rRNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specimens using the C. trachomatis‐specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other bacterial pathogens from the same PCR product by varying the hybridization probes.