Sulfonamide resistance in Haemophilus influenzae mediated by acquisition of sul2 or a short insertion in chromosomal folP

Determinants of sulfonamide resistance were investigated in clinical isolates of Haemophilus influenzae from the United Kingdom and Kenya. The mechanism of sulfonamide resistance in H. influenzae has not previously been reported. Eight isolates requiring at least 1,024 microg of sulfamethoxazole per ml for inhibition carried the sul2 gene, a common mediator of acquired sulfonamide resistance in enteric bacteria. In other isolates with similarly high levels of resistance, the chromosomal gene encoding dihydropteroate synthase, folP, was found to carry an insertion of 15 bp together with other missense mutations relative to folP of H. influenzae strain Rd RM118 (MIC, 8 microg/ml); the folP sequence was identical in all seven such isolates investigated, although they represented three different strains by restriction pattern analysis. The 15-bp insertion was absent in isolates inhibited by sulfamethoxazole at 2 to 64 microg/ml (although these exhibited considerable divergence in folP sequence) and in highly resistant isolates carrying sul2. Transformation with a 599-bp fragment of folP containing the insertion but no other differences conferred high-level resistance on a recipient strain, confirming the role of the insertion. Other amino acid substitutions in dihydropteroate synthase may modulate the level of sulfonamide inhibition in susceptible isolates and those with more moderate levels of resistance. The two mechanisms of resistance, mediated by sul2 and modified folP, were detected in isolates from both the United Kingdom and Kenya.     

Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure

OBJECTIVES: Despite a 97% reduction in clinical sulphonamide usage, the prevalence of sulphonamide resistance among Escherichia coli has remained constant in the UK. Genetic linkage of sulphonamide resistance to other resistances is thought important for this maintenance, but the finding also implies that sulphonamide resistance exerts little fitness cost. To test this hypothesis, we examined the fitness impact of four naturally occurring sul2-coding plasmids upon their hosts. METHODS: The fitness impact of the plasmids upon E. coli was determined by pairwise growth competition in a minimal medium. The DNA sequence of plasmid p9123 was obtained by primer walking and PCR. RESULTS: Three of the four sul2-coding plasmids studied imposed fitness costs on their hosts. The fourth plasmid, a 6.2 kb resistance element carrying sul2, strA and strB designated p9123, conferred a 4% fitness advantage upon its original clinical host and also on E. coli K12 JM109. The complete sequence of p9123 revealed eight open reading frames, including five of unknown function. There was no obvious gene to which the fitness advantage might be attributed. CONCLUSIONS: The novel finding that p9123 can improve host fitness may explain why this plasmid and its close relatives are so widespread among enteric bacteria. In addition to other factors such as co-selection of sulphonamide resistance by other agents, the fitness advantage conferred by plasmids such as p9123 may have contributed to the maintenance of sulphonamide resistance in the UK in the absence of clinical selection pressure. These data indicate that once antibiotic resistance has been established on mobile genetic elements, it may be difficult to eliminate.     

Prevalence of adrenal haemorrhage in non-surviving patients with burns

Purpose

The purpose of this study was to investigate the prevalence of adrenal haemorrhage (AH) in non-surviving patients with severe burns by combining the clinical notes and autopsy data.

Methods

A retrospective review of all adult (age >18 years) burn patients who died in the Helsinki Burn Centre, Helsinki, Finland during 1.1.1995 to 31.12.2005 was conducted. Patients diagnosed with AH either in the clinical charts or in the autopsy reports were sorted out. Special attention was paid to the course of events preceding death and autopsy findings.

Results

We identified four patients, creating a prevalence of 5.6%. None of the patients was diagnosed with AH alive. Three patients we diagnosed with bilateral AH, all young men. Cause of death was multiple organ failure (MOF) in all three cases. On the autopsy, they were diagnosed with 5 ± 1 organ failures. One case of unilateral AH was established, an elderly male with hot air sauna burns. Clinical charts and autopsy data suggest idiopathic AH.

Conclusions

The prevalence of AH is higher than previously estimated in non-surviving patients with burns. Bilateral AH occurred later then 1 week after the burn trauma. Bilateral AH always presented with sepsis, multiple organ failure and acute renal failure.     

Recurrent DNA copy number changes revealed by comparative genomic hybridization in primary Merkel cell carcinomas.

Comparative genomic hybridization (CGH) was used to search for gains, high-level amplifications and losses of DNA sequences along all chromosome arms in 19 primary Merkel cell carcinomas (MCC). Extensive genetic aberrations, with a mean value of 5.5+/-1.1 changes per tumor were detected in 13 out of the 19 samples analyzed. Our CGH results reveal several new and other previously known chromosomal regions that are involved in the pathogenesis of MCC. The majority of the alterations were gains of whole chromosomes or whole chromosome arms. Compared to losses, the frequency of DNA copy number gains was two-fold. DNA sequence copy number gains were most common in chromosomes 6 (42%), 1 (37%), and 5 (32%). The most frequent minimal common regions of gains were 6pterqter (42%), 1q11q31 (32%), and 5p (32%). No recurrent high-level amplifications were observed. High-level amplifications of small chromosomal regions were found in four samples out of the 19 tumors analyzed (21%). Amplifications affected 1q22q24 (5%), 4p (5%), and 5p (5%). Losses most frequently affected chromosomes 13 (21%) and 4 (16%). Minimal common regions with the most frequent losses were 13q13q31 (21%), 4q (16%), and 16q (11%). No significant statistical correlation between genomic aberrations and clinicopathological factors was revealed, despite the fact that there was an obvious tendency towards it. Primary MCC expressing DNA alterations were predominantly distinguished in large tumors, and risk of metastatic dissemination was three-fold compared to tumors with no DNA alterations.     

Mesh-based method for measuring intracranial volume in patients with craniosynostosis

Purpose

   Craniosynostosis may lead to reduced intracranial volume (ICV) and disturb normal brain growth and development. Thus, ICV is an important parameter with respect to the surgical outcome. Current methods for ICV determination from computed tomography (CT) images have drawbacks. The aim of this study was to investigate the performance of the novel mesh-based method (MBM) for ICV determination with craniosynostosis patients.

Methods

   Twenty-two patients operated on for scaphocephaly were included in this study. ICVs from preoperative, one-week postoperative, and one-year postoperative CT images were measured with MBM. The level of agreement with the manual segmentation method (MSM) was determined for the measurements of preoperative and one-year postoperative datasets. Repeatability was determined with re-measurements of six datasets. Measurement time was recorded for MBM.

Results

   Mean $(\pm \text{ SD})$ preoperative ICV values were 895.0 $\pm $ 153.1 $\text{ cm}^{3}$ and 896.4 $\pm $ 147.2 $\text{ cm}^{3}$ as measured with MBM and MSM, respectively. Corresponding one-year postoperative values were 1,238.3 $\pm $ 118.7 $\text{ cm}^{3}$ and 1,250.1 $\pm $ 117.5 $\text{ cm}^{3}$ . The MBM allowed ICV determination from one-week postoperative datasets. Measurement time with MBM was 4

Conclusions

   MBM is an efficient method for determining the ICV of craniosynostosis patients, allowing the measurement of skulls with bony defects. The repeatability and short measurement time of MBM are attributable to the user interference and assessment of the measurement process.     

FK506 blocks activation of the intrinsic caspase cascade after optic nerve crush

Retinal ganglion cells die by apoptosis after optic nerve crush. FK506 has been shown to be neuroprotective in this model but the mechanism(s) by which it exerts these actions remains unknown. We and others have shown that caspase 9 is cleaved in the retina in other injury models and we hypothesized that the neuroprotection observed with FK506 was mediated by interference with caspase 9 activation. The present study examined the cellular localization of caspase 9 cleavage after intraorbital optic nerve crush in rats, the time course of caspase 9 cleavage after optic nerve crush and the ability of orally administered FK506 to block caspase 9 cleavage after optic nerve crush. We show by immunohistochemistry that cleaved caspase 9 is present in retinal ganglion cells (identified by prior backlabelling) after optic nerve crush. Immunoblot analysis showed that caspase 9 cleavage is significantly elevated 5 and 8 days after optic nerve crush. We show that orally administered FK506 reaches the retina and is pharmacologically active in retinal tissue. Furthermore, the oral administration of FK506 5 mg kg(-1) day(-1) blocks the cleavage of caspase 9 at both time points. These data suggest that caspase 9 activation may play an important role in retinal ganglion cell death following optic nerve crush and that the neuroprotection seen with FK506 may be mediated by interfering with the activation of caspase 9.