To Örebro University

Objective: To survey the placental gene expression of inflammatory markers and growth factors in non-smoking obese women with an uncomplicated pregnancy without associated morbidity and delivery at term compared with normal weight women.

Methods: Placental tissue samples from 32 obese women (body mass index, BMI >= 35.0 kg/m(2)) were compared with samples from 94 normal weight women (BMI 18.5-25.0 kg/m(2)) matched for age (+/- 1 year), gestational age (+/- 3 days), parity and mode of delivery. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to analyse toll receptor-2 and -4, interleukin-6 and -8, tumour necrosis factor-alpha, leptin, adiponectin, insulin-like growth factor-1 and -2, hepatocyte growth factor, hepatocyte growth factor receptor and insulin receptor.

Results: There was no significant difference in gene expression in placental tissue samples from obese and normal weight women.

Conclusion: We found no difference in the occurrence of inflammatory marker and growth factor mRNA levels in placental tissue samples from a large group of obese women without associated morbidity and with healthy infants compared to a closely matched control group of healthy normal weight women. Compared with the previous studies, this anomalous finding may be explained by the absence of associated morbidity in the obese women in our study.

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 1-36h. 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 36h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of - and -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)- 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- 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.

Aim: 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.

Methods: Human vascular endothelial cells and small airway epithelial cells were incubated with neonatal blood isolates of Staphylococcus epidermidis (n = 14) and Staphylococcus aureus (n = 14). The extracellular release of IL-8, IL-10, sICAM-1, ICAM-1 mRNA and the expression of membrane bound ICAM-1 were assessed by ELISA, RT-PCR and immunofluorescence microscopy.

Results: Staphylococcus epidermidis induced higher levels of IL-8 (mean 38.5 ng/mL) and ICAM-1 mRNA (mean ratio 1.037) in the small airway epithelial cells than S. aureus (IL-8 mean 22.2 ng/mL, p < 0.01 and ICAM-1 mRNA mean ratio 0.715, p < 0.01). In the endothelial cells, ICAM-1 remained more integrated in the cell membranes after exposure to S. epidermidis compared with S. aureus, which induced disintegration and release of soluble ICAM-1 into the supernatants.

Conclusion: Staphylococcus epidermidis induced a higher chemoattractive response than S. aureus. A persistent transmigration of granulocytes into the lung tissue in neonatal S. epidermidis sepsis might contribute to the development of bronchopulmonary dysplasia.

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.

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.

Methods: Among 401 newborn infants <28 days of age with suspected sepsis, nine signs of sepsis and C-reactive protein (CRP) values were prospectively recorded. Logistic regression assessed the association of these signs and laboratory values with a subsequently confirmed diagnosis of sepsis by positive blood culture. The analysis was stratified by gestational age with mutual simultaneous adjustment for the signs and sex.

Results: Five of the nine clinical signs (feeding intolerance, distended abdomen, blood pressure, bradycardia and apnoea), along with CRP were statistically significantly associated with a positive blood culture. After simultaneous adjustment for all of the signs, apnoea, hypotension and CRP were independently predictive of positive blood culture. When the material was stratified by gestational age, differences in the association with positive blood culture were found for bradycardia, tachypnea and irritability/seizures.

Conclusion: In this selected population of infants with suspected sepsis, apnoea and hypotension are independently predictive of a confirmed diagnosis, while bradycardia is more predictive among preterm infants and tachypnea among term infants.

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.

Staphylococcus epidermidis is a significant pathogen in neonatal sepsis and other nosocomial infections. For further investigations of the colonisation patterns and invasive pathways, typing methods that are applicable on large populations of bacterial isolates are warranted. In the present study, a genotyping method based on polymerase chain reaction (PCR) for the repeat regions of four genes (sdrG, sdrF, aap and sesE) that encode for bacterial surface proteins was developed and applied to a sample of well-characterised neonatal blood isolates of S. epidermidis (n = 49). The PCR products were visualised on agarose gel (sdrG, sdrF and sesE) or by fragment analysis (aap). The discriminatory index (D-index) for genotyping of the different genes was compared to genotyping by pulsed-field gel electrophoresis (PFGE). The highest D-index for the PCR-based typing methods was found for the combination of sdrF, sdrG and aap (D-index 0.94), whereas the optimal two-gene combination (sdrF and aap) resulted in a D-index of 0.92. We conclude that the described method can be used for the genotyping of large populations of S. epidermidis isolates with a sufficient discriminatory capacity, and we suggest that the combination of sdrF and aap is the most suitable to use.

OBJECTIVE: To evaluate a real-time PCR assay for the diagnosis of neonatal bacteraemia. PATIENTS AND METHODS: Two hundred ninety-five plasma samples from 288 newborns with suspected neonatal sepsis were collected prospectively for the purpose of polymerase chain reaction (PCR)-based bacterial detection. A real-time PCR targeting the bacterial gene for 16S-rRNA gene combined with four specific probes designed to detect Gram-negative bacteria, Staphylococcus aureus and coagulase-negative staphylococci (CoNS) was developed. All samples positive in the universal PCR were further sequenced for bacterial identification. RESULTS: When applied to a material from 50 patients with positive blood culture and 245 patients with negative blood culture, the universal PCR showed a sensitivity of 42% (28-57), a specificity of 95% (92-97), a positive predictive value of 64% (45-80), and a negative predictive value of 89% (84-92) (95% confidence intervals in brackets). CONCLUSION: A new real-time PCR technique was for the first time applied to a well-defined prospectively and consecutively enrolled material of newborns with suspected sepsis, combining the benefits of real-time PCR with specific probes and sequencing. The method managed to detect bacteraemia with high specificity even though the sensitivity was low. Factors causing the low sensitivity are identified and further strategies to develop the method are described.