Maria Björkqvist

Phenotypic and genotypic characterisation of blood isolates of coagulase‐negative staphylococci in the newborn

Coagulase‐negative staphylococci (CNS) are the leading cause of late‐onset sepsis in newborns (>72 h of age). Our aim was to determine whether phenotypic and/or genotypic differences existed between blood isolates of CNS regarded as inducers of sepsis or as contaminants. Ninety‐seven bloodisolates of CNS recovered from newborns at the neonatal intensive care unit, Örebro, Sweden in 1983–1997 were analysed. Twenty‐nine of them (30%) were classified as sepsis isolates and 68 (70%) as contaminants. The most prevalent species was Staphylococcus epidermidis (n=59). Staphylococcus haemolyticus (n=16) was most often isolated from newborns with the lowest gestational age and birth weight. Biochemical typing using the Phene Plate system (PhP) and genotyping using pulsed‐field gel electrophoresis (PFGE) showed that the S. epidermidis isolates regarded as inducers of sepsis (n=16) were more homogeneous than isolates considered contaminants (n=37). One main genotypic group, representing seven (44%) isolates, was identified among the sepsis isolates. Phenotypically the S. epidermidis sepsis isolates comprised three major clusters. In contrast, among the S. epidermidis contaminants, eight genotypic groups and two phenotypic clusters were identified. The dominating genotypic group among the sepsis isolates of S. epidermidis may represent strains with higher invasive capacity.

Defective Neutrophil Oxidative Burst in Preterm Newborns on Exposure to Coagulase-Negative Staphylococci

The neutrophil oxidative burst is a product of the regulated assembly of the multicomponent oxidase enzyme. Our aim was to compare the oxidative burst in term (n = 10) and preterm newborns <31 wk gestational age (n = 10) after stimulation with coagulase-negative staphylococci in vitro. Strains of Streptococcus epidermidis with different invasive and slime-producing properties, one strain of S. haemolyticus, and one strain of group B-streptococcus were investigated. A whole-blood flow cytometric assay using the oxidation of hydroethidine to ethidium bromide was used. The oxidative activity in unstimulated neutrophil granulocytes [polymorphonuclear leukocytes (PMNLs)] was similar in term and preterm newborns, but the preterm newborns showed a significantly lower capacity to up-regulate the oxidative burst intensity after bacterial stimulation (p = 0.004). In the term but not in the preterm group, the oxidative burst intensity after bacterial stimulation correlated with the baseline oxidative burst intensity. After bacterial stimulation, there was a trend toward a greater percentage of activated neutrophils in the term group than in the preterm group, but the difference was less pronounced than that in oxidative burst intensity. Significant differences in oxidative burst response to different bacterial strains were observed (p < 0.001), but the differences could not be correlated exclusively to invasive capacity or slime-producing properties. It is concluded that the baseline oxidative activity is similar in term and preterm PMNLs but that preterm PMNLs have a decreased capacity to increase the oxidative burst in response to bacterial stimulation.

Real‐time PCR of the 16S‐rRNA gene in the diagnosis of neonatal bacteraemia

Objective: To evaluate a real‐time PCR assay for the diagnosis of neonatal bacteraemia.

Human neutrophil lipocalin: normal levels and use as a marker for invasive infection in the newborn.

Aim: To evaluate human neutrophil lipocalin (HNL) as a marker of neonatal invasive infection and determine the normal serum levels of HNL in newborns. Methods: HNL is released from neutrophil granulocytes and is regarded as a specific marker of neutrophil activity. In 81 newborns 28 d of age with signs of infection on a total of 87 occasions, HNL and C‐reactive protein (CRP) were measured at inclusion and on the three following days. As controls, term healthy newborns were recruited at birth (cord blood, n= 45) and at ages 3‐5 d (n= 46). Serum HNL was measured by a radioimmunoassay. Results: 25/87 episodes were classified as infection and 62 as non‐proven infection. HNLmax was significantly higher in the infected group (mean 587.6 μg/1) than in the non‐proven infected group (mean 217.7 μg/1, p > 0.001). HNL peaked at inclusion, 1 d earlier than CRP. In the healthy controls, HNL was the same at 3‐5 d of age as at birth (mean 82.4‐81.7 μg/1) and similar to normal adult levels.

Bottle-blowing in hospital-treated patients with community-acquired pneumonia.

A study was carried out to determine whether bottle-blowing has any positive effects in patients with pneumonia. In a prospective open study 145 adults with untreated community-acquired pneumonia requiring hospitalization were randomized to early mobilization (group A), to sit up and take 20 deep breaths on 10 occasions daily (group B), or to sit up and to blow bubbles in a bottle containing 10 cm water through a plastic tube 20 times on 10 occasions daily (group C). Peak expiratory flow (PEF), vital capacity (VC), forced expiratory volume in 1 sec (FEV1) and serum concentration of C-reactive protein (CRP) were determined on admission, and on days 4 and 42. Fever duration and hospital stay were recorded. In a subset of 16 patients, single breath diffusion capacity of carbon monoxide was measured on 3 occasions. The patients in group A were hospitalized for a mean of 5.3 days, group B for 4.6 days and group C for 3.9 days. Treatment was a significant factor (p = 0.037) in a Cox regression model, with group C significantly better than group A (p = 0.01). The number of days with fever was 2.3, 1.7 and 1.6 in groups A, B and C respectively. These differences were not significant (p = 0.28). No significant differences were found between the groups regarding CRP, PEF, VC, FEV1, or diffusion capacity. Intensive bottle-blowing shortens the hospital stay in patients with pneumonia. The underlying mechanism is not clear.

Diagnosis of neonatal sepsis by broad-range 16S real-time polymerase chain reaction.

Background: The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity and specificity, and 16S polymerase chain reaction (PCR) has been suggested as a new diagnostic tool for neonatal sepsis. Objectives: To develop and evaluate a new real-time PCR method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Methods: Immediately after blood culture, a study sample of 0.5–1.0 ml whole blood was collected and used for a novel 16S real-time PCR assay. All positive samples were sequenced. Detailed case studies were performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis. Results: 368 samples from 317 infants were included. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. Seven of the 31 samples with a positive PCR result and a negative blood culture had definite or suspected bacterial sepsis. In five samples, PCR (but not blood culture) could detect a pathogen that was present in a blood culture collected more than 24 h prior to the PCR sample. Conclusions: This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even after the initiation of intravenous antibiotics.

Colonization pattern of coagulase-negative staphylococci in preterm neonates and the relation to bacteremia

Coagulase-negative staphylococci (CoNS) are the major cause of sepsis in extreme preterm (EPT) newborns, but data on the CoNS colonization in EPT newborns prior to invasive infection are limited. Our aim was to describe the early establishment of the CoNS microflora in EPT newborns and to compare the colonization pattern in neonates with and without positive CoNS blood cultures. From a cohort of 46 EPT neonates, newborns with positive CoNS blood culture were identified (n = 10) and compared with matched controls. Samples for bacterial cultures were obtained repetitively from nares, perineum, and umbilicus. All CoNS isolates were characterized using the PhenePlate system for biochemical fingerprinting. Persistent CoNS strains were found on day 2–3 after delivery in 7/20 newborns, and there was a tendency for earlier colonization in nares than in the perineum or umbilicus. The CoNS blood strains were prevalent in superficial sites prior to positive blood culture (11/14 blood strains), but no single invasive pathway was identified. Most CoNS blood strains (9/14) persisted on superficial sites after antibiotic treatment. We hypothesize that the invasive pathways in neonatal CoNS sepsis are complex and that the colonization of mucosal membranes and umbilical catheters might be of equal importance.

Clinical signs and CRP values associated with blood culture results in neonates evaluated for suspected sepsis.

Aim:  To identify which clinical signs at presentation are most predictive of sepsis subsequently confirmed by blood culture and to investigate whether the predictive power of the clinical signs varies by gestational age.

Modulation of ENaC, CFTR, and iNOS expression in bronchial epithelial cells after stimulation with Staphylococcus epidermidis (94B080) and Staphylococcus aureus (90B083).

Bacteria affect the respiratory epithelium, which is covered by airway surface liquid (ASL) and mucus. Ion concentrations in the ASL are determined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC). Neonatal sepsis is a major risk factor for subsequent pulmonary disease in preterm newborns. Predominating are coagulase‐negative staphylococci (e.g., Staphylococccus epidermidis and Staphylococccus aureus). The aim of this study was to investigate modulation of CFTR, ENaC, mucins, proinflammatory cytokines, and inducible nitric oxide synthase (iNOS) in respiratory epithelial cells after S. epidermidis 94B080 and S. aureus 90B083 exposure. Bronchial epithelial cells were incubated with S. epidermidis 94B080 and S. aureus 90B083 (neonatal blood isolates) for 1–36 h. Expression of CFTR, ENaC, iNOS, and mucins was analyzed by real‐time PCR and Western blotting. Release of cytokines was analyzed by ELISA, and production of NO by the Griess assay. Expression of CFTR significantly decreased after 36 h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of β‐ and γ‐ENaC. Expression of iNOS increased, but NO was not detected. Both staphylococci caused a decrease in the intracellular Ca2+ concentration. S. aureus induced increased secretion of IL‐6, IL‐8, and transforming nuclear factor (TNF)‐α in a time‐dependent manner as compared with S. epidermidis. In conclusion, expression of ENaC, CFTR, and iNOS is modulated by exposure to S. aureus 90B083 and S. epidermidis 94B080. S. aureus is more potent in causing release of IL‐6, IL‐8, and TNF‐α by bronchial epithelial cells as compared with S. epidermidis. The mRNA expression for the mucus proteins MUC2, MUC5AC, and MUC5B could not be measured, neither in the presence nor in the absence of bacteria.

Staphylococcus epidermidis and Staphylococcus aureus trigger different interleukin-8 and intercellular adhesion molecule-1 in lung cells: implications for inflammatory complications following neonatal sepsis

Staphylococci are a major contribution for neonatal sepsis, which is the main risk factor for bronchopulmonary dysplasia. This study investigated the expression of pro‐inflammatory mediators in endothelial and respiratory cells from newborns exposed to staphylococci.