Claus Klingenberg

Cranioectodermal Dysplasia, Sensenbrenner Syndrome, Is a Ciliopathy Caused by Mutations in the IFT122 Gene

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.

Biofilm Formation by Staphylococcus haemolyticus

ABSTRACT Infections due to coagulase-negative staphylococci (CoNS) most frequently occur after the implantation of medical devices and are attributed to the biofilm-forming potential of CoNS. Staphylococcus haemolyticus is the second most frequently isolated CoNS from patients with hospital-acquired infections. There is only limited knowledge of the nature of S. haemolyticus biofilms. The aim of this study was to characterize S. haemolyticus biofilm formation. We analyzed the biofilm-forming capacities of 72 clinical S. haemolyticus isolates. A detachment assay with NaIO4, proteinase K, or DNase was used to determine the main biofilm components. Biofilm-associated genes, including the ica operon, were analyzed by PCR, and the gene products were sequenced. Confocal laser scanning microscopy (CLSM) was used to elucidate the biofilm structure. Fifty-three isolates (74%) produced biofilms after growth in Trypticase soy broth (TSB) with glucose, but only 22 (31%) produced biofilms after growth in TSB with NaCl. It was necessary to dissolve the biofilm in ethanol-acetone to measure the optical density of the full biofilm mass. DNase, proteinase K, and NaIO4 caused biofilm detachment for 100%, 98%, and 38% of the isolates, respectively. icaRADBC and polysaccharide intercellular adhesin (PIA) production were found in only two isolates. CLSM indicated that the biofilm structure of S. haemolyticus clearly differs from that of S. epidermidis. We conclude that biofilm formation is a common phenotype in clinical S. haemolyticus isolates. In contrast to S. epidermidis, proteins and extracellular DNA are of functional relevance for biofilm accumulation, whereas PIA plays only a minor role. The induction of biofilm formation and determination of the biofilm mass also needed to be optimized for S. haemolyticus.

Coagulase-negative staphylococcal sepsis in neonates. Association between antibiotic resistance, biofilm formation and the host inflammatory response.

Background: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late onset sepsis in neonates. They are often multiresistant to antibiotics, and the ability to form biofilm is considered their main virulence determinant. Methods: During a 12-year period, we identified 150 neonates having 164 suspected septic episodes with growth of CoNS in blood culture. We examined the relationship between antibiotic resistance, phenotypic biofilm production and genetic determinants for biofilm formation in different CoNS species and their correlation with neonatal inflammatory response. Results: Eighty-five episodes were classified as true sepsis, and 79 episodes of CoNS growth in blood culture were considered contaminations. Sixty-one percent of Staphylococcus epidermidis isolates produced biofilm compared with 26% of CoNS non-epidermidis (P < 0.001). We observed no difference in phenotypic biofilm production or genetic determinants for biofilm formation between invasive isolates and contaminants. C-reactive protein levels as a marker of inflammatory response were higher in CoNS sepsis caused by methicillin and aminoglycoside resistant versus susceptible isolates (P = 0.031). In contrast, there was a significant association between a lower C-reactive protein response and biofilm-positive isolates (P = 0.018). Antibiotic resistance was significantly correlated with biofilm production in S. epidermidis, but not in other CoNS species. Conclusions: CoNS sepsis with biofilm-forming strains was associated with a decreased host inflammatory response, potentially limiting the immune system to counteract the infection. The impact of antibiotic resistance and virulence determinants on clinical outcome of neonatal CoNS sepsis warrants additional clinical studies.

Volume-Targeted versus Pressure-Limited Ventilation for Preterm Infants: A Systematic Review and Meta-Analysis

Background: The causes of bronchopulmonary dysplasia (BPD) are multifactorial. Overdistension of the lung (volutrauma) is considered an important contribution. As an alternative to traditional pressure-limited ventilation (PLV), modern neonatal ventilators offer modes which can target a set tidal volume. Objectives: To determine whether volume-targeted neonatal ventilation, compared with PLV, reduces death or BPD. Methods: We performed a systematic review and meta-analysis using the methodology of the Neonatal Review Group of the Cochrane Collaboration. A comprehensive literature search was undertaken, and data for prespecified outcomes were combined where appropriate using the fixed effects model. Results: Nine trials were eligible. Volume-targeted ventilation resulted in a reduction in: the combined outcome of death or BPD [typical relative risk, RR, 0.73 (95% confidence interval, 0.57–0.93), numbers needed to treat, NNT, 8 (95% CI 5–33)], the incidence of pneumothorax [typical RR 0.46 (95% CI 0.25–0.84), NNT 17 (95% CI 10–100)], days of ventilation [weighted mean difference 0.8 days (log-transformed data, p = 0.05)], hypocarbia (pCO2 <35 mm Hg/4.7 kPa); [typical RR 0.56 (95% CI 0.33–0.96), NNT 4 (95% CI 2–25)], and the combined outcome of periventricular leukomalacia or grade 3–4 intraventricular hemorrhage [typical RR 0.48 (95% CI 0.28–0.84), NNT 11 (95% CI 7–50)]. Conclusions: Compared with PLV, infants ventilated using volume-targeted ventilation had reduced death/BPD, duration of ventilation, pneumothoraces, hypocarbia and periventricular leukomalacia/severe intraventricular hemorrhage. Further studies are needed to assess neurodevelopmental outcomes.

Effect of sustained inflation duration; resuscitation of near-term asphyxiated lambs

Objective The 2010 ILCOR neonatal resuscitation guidelines do not specify appropriate inflation times for the initial lung inflations in apnoeic newborn infants. The authors compared three ventilation strategies immediately after delivery in asphyxiated newborn lambs. Design Experimental animal study. Setting Facility for animal research. Subjects Eighteen near-term lambs (weight 3.5–3.9 kg) delivered by caesarean section. Interventions Asphyxia was induced by occluding the umbilical cord and delaying ventilation onset (10–11 min) until mean carotid blood pressure (CBP) was ≤22 mm Hg. Animals were divided into three groups (n=6) and ventilation started with: (1) inflation times of 0.5 s at a ventilation rate 60/min, (2) five 3 s inflations or (3) a single 30 s inflation. Subsequent ventilation used inflations at 0.5 s at 60/min for all groups. Main outcome measures Times to reach a heart rate (HR) of 120 bpm and a mean CBP of 40 mm Hg. Secondary outcome was change in lung compliance. Results Median time to reach HR 120 bpm and mean CBP 40 mm Hg was significantly shorter in the single 30 s inflation group (8 s and 74 s) versus the 5×3 s inflation group (38 s and 466 s) and the conventional ventilation group (64 s and 264 s). Lung compliance was significantly better in the single 30 s inflation group. Conclusion A single sustained inflation of 30 s immediately after birth improved speed of circulatory recovery and lung compliance in near-term asphyxiated lambs. This approach for neonatal resuscitation merits further investigation.

High in vitro antimicrobial activity of synthetic antimicrobial peptidomimetics against staphylococcal biofilms

OBJECTIVES The aim of the study was to investigate the antimicrobial effect of different antibiotics and synthetic antimicrobial peptidomimetics (SAMPs) on staphylococcal biofilms. METHODS Biofilms of six staphylococcal strains (two Staphylococcus haemolyticus, two Staphylococcus epidermidis and two Staphylococcus aureus isolates) were grown for 24 h in microtitre plates. They were washed and treated for 24 h with different concentrations of linezolid, tetracycline, rifampicin and vancomycin and four different SAMPs. After treatment, the redox indicator Alamar Blue was used to quantify metabolic activity of bacteria in biofilms, and confocal laser scanning microscopy with LIVE/DEAD staining was used to further elucidate any effects. RESULTS At MIC levels, rifampicin and tetracycline showed a marked reduction of metabolic activity in the S. epidermidis and S. haemolyticus biofilm. Linezolid had a moderate effect and vancomycin had a poor effect. MIC x10 and MIC x100 improved the antimicrobial activity of all antibiotics, especially vancomycin. However, metabolic activity was not completely suppressed in strong biofilm-producing strains. At MIC x10, the three most effective SAMPs (Ltx5, Ltx9 and Ltx10) were able to completely eliminate metabolic activity in the S. epidermidis and S. haemolyticus biofilms, which was also confirmed by complete cell death using confocal laser scanning microscopy investigations. Although none of the Ltx SAMPs could fully suppress metabolic activity in the S. aureus biofilm, their effect was superior to all tested antibiotics. CONCLUSIONS SAMPs had superior antimicrobial activity in staphylococcal biofilms compared with conventional antibiotics and are potential new therapeutic agents for biofilm-associated infections.

Patient comfort during treatment with heated humidified high flow nasal cannulae versus nasal continuous positive airway pressure: a randomised cross-over trial

Objective To compare patient comfort in preterm infants treated with heated humidified high flow nasal cannulae (HHHFNC) versus nasal continuous positive airway pressure (NCPAP). Design Randomised cross-over trial (2×24 h). Setting Single tertiary neonatal unit. Patients 20 infants less than 34 weeks postmenstrual age treated with NCPAP due to mild respiratory illness. Interventions After parental consent, infants were randomised to 24 h of treatment with NCPAP or HHHFNC followed by 24 h of the alternate therapy. Main outcome measures Primary outcome was patient comfort assessed by the EDIN (neonatal pain and discomfort) scale. Secondary outcomes were respiratory parameters (respiratory rate, FiO2, SpO2, TcPCO2), ambient noise, salivary cortisol and parental assessments of their child. Results We found no differences between HHHFNC and NCPAP in mean cumulative EDIN score (10.7 vs 11.1, p=0.25) or ambient noise (70 vs 74 dBa, p=0.18). Parents assessed HHHFNC treatment as significantly better in the three domains, 1) child satisfied, 2) parental contact and interaction and 3) possibility to take part in care. Mean respiratory rate over 24 h was lower during HHHFNC than CPAP (41 vs 46, p=0.001). Other respiratory parameters were similar. Conclusions Using EDIN scale, we found no difference in patient comfort with HHHFNC versus NCPAP. However, parents preferred HHHFNC, and during HHHFNC respiratory rate was lower than during NCPAP. ClinicalTrials.gov, number NCT01526226.

Expired CO2 Levels Indicate Degree of Lung Aeration at Birth

As neonatal resuscitation critically depends upon lung aeration at birth, knowledge of the progression of this process is required to guide ongoing care. We investigated whether expired CO2 (ECO2) levels indicate the degree of lung aeration immediately after birth in two animal models and in preterm infants. Lambs were delivered by caesarean section and ventilated from birth. In lambs, ECO2 levels were significantly (p<0.0001) related to tidal volumes and CO2 clearance/breath increased exponentially when tidal volumes were greater than 6 mL/kg. Preterm (28 days of gestation; term = 32 days) rabbits were also delivered by caesarean section and lung aeration was measured using phase contrast X-ray imaging. In rabbit kittens, ECO2 levels were closely related (p<0.001) to lung volumes at end-inflation and were first detected when ∼7% of the distal lung regions were aerated. ECO2 levels in preterm infants at birth also correlated with tidal volumes. In each infant, ECO2 levels increased to >10 mmHg 28 (median) (21–36) seconds before the heart rate increased above 100 beats per minute. These data demonstrate that ECO2 levels can indicate the relative degree of lung aeration after birth and can be used to clinically assess ventilation in the immediate newborn period.

Sustained Inflations: Comparing Three Neonatal Resuscitation Devices

Background: Some national resuscitation guidelines advocate using sustained initial inflations (2–3 s) for babies requiring resuscitation. Inflation times ≧10 s have been used for preterm infants. Objectives: This study examines the ability of operators of varying experience to provide a sustained inflation using three different manual ventilation devices. Methods: We compared a self-inflating bag, a flow-inflating bag and a pressure-limited T-piece device. Fifty clinical staff members from five professional groups gave a sustained inflation with a target peak pressure of 30 cm H2O and target duration of 10 s to an internal leak-free manikin. We measured peak inflating pressure (PIP) and mean inflating pressure (MIP) during the sustained inflation, and the duration of inflating pressure (IP) >20 and 25 cm H2O. Results: Median (IQR) duration of IP >25 cm H2O was: self-inflating bag 2.5 s (0.8–5.7), flow-inflating bag 10.6 s (8.4–12.9) and the T-piece 10.7 s (8.9–11.9). There was a weak correlation between experience using a self-inflating bag and longer inflation times (R = 0.290, p = 0.041). When compared with the T-piece, the flow-inflating bag had lower mean MIP (27.0 ± 1.8 vs. 28.8 ± 2.0 cm H2O) and higher mean PIP (32.3 ± 3.7 vs. 29.8 ± 1.8 cm H2O). There were no differences in performance between operator groups. Conclusion: The T-piece provided consistent PIP during a single 10 s sustained inflation with less variation in pressure compared with the flow-inflating bag. Sustained inflations >3 s were difficult to achieve with a self-inflating bag.

Single Sustained Inflation followed by Ventilation Leads to Rapid Cardiorespiratory Recovery but Causes Cerebral Vascular Leakage in Asphyxiated Near-Term Lambs

Background A sustained inflation (SI) rapidly restores cardiac function in asphyxic, bradycardic newborns but its effects on cerebral haemodynamics and brain injury are unknown. We determined the effect of different SI strategies on carotid blood flow (CaBF) and cerebral vascular integrity in asphyxiated near-term lambs. Methods Lambs were instrumented and delivered at 139 ± 2 d gestation and asphyxia was induced by delaying ventilation onset. Lambs were randomised to receive 5 consecutive 3 s SI (multiple SI; n = 6), a single 30 s SI (single SI; n = 6) or conventional ventilation (no SI; n = 6). Ventilation continued for 30 min in all lambs while CaBF and respiratory function parameters were recorded. Brains were assessed for gross histopathology and vascular leakage. Results CaBF increased more rapidly and to a greater extent during a single SI (p = 0.01), which then decreased below both other groups by 10 min, due to a higher cerebral oxygen delivery (p = 0.01). Blood brain barrier disruption was increased in single SI lambs as indicated by increased numbers of blood vessel profiles with plasma protein extravasation (p = 0.001) in the cerebral cortex. There were no differences in CaBF or cerebral oxygen delivery between the multiple SI and no SI lambs. Conclusions Ventilation with an initial single 30 s SI improves circulatory recovery, but is associated with greater disruption of blood brain barrier function, which may exacerbate brain injury suffered by asphyxiated newborns. This injury may occur as a direct result of the initial SI or to the higher tidal volumes delivered during subsequent ventilation.