Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone

To study the epidemiology of Pseudomonas aeruginosa colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental P. aeruginosa isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with P. aeruginosa, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal P. aeruginosa colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD(r)). The MDR strain was found at a higher frequency in sputum samples than the SSD(r) strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (oprI, oprL, and oprD) and the pyoverdine type revealed that all predominant strains except the SSD(r) strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.     

Percoll gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm injection

Prior to intracytoplasmic sperm injection (ICSI), seminal fluid is currently washed out from the ejaculated semen and further sperm selection is carried out by a discontinuous Percoll gradient. Possible deleterious effects from the sperm-separating substance Percoll on sperm function or embryo cleavage after in-vitro fertilization (IVF) have, to our knowledge, not yet been reported and the use of Percoll has been widely accepted in IVF. In this study, we examined whether the omission of the Percoll step in the sperm preparation has any influence on the outcome of the ICSI procedure. Two methods of sperm preparation for ICSI were compared in a controlled study on sibling oocytes: washing the semen sample once, followed by a Percoll gradient centrifugation versus washing the sperm sample twice without a Percoll gradient centrifugation. The mean fertilization rates were similar for the two sperm preparation methods: 78.2 +/- 21.4 and 75.0 +/- 24.1% respectively of the intact oocytes displaying two pronuclei. Cleavage rates did not differ statistically between the two groups. Whereas in both groups similar percentages of excellent, good and poor quality embryos were obtained, the percentage of fair quality embryos was significantly higher in the group without Percoll (16.3 +/- 20.1 versus 9.1 +/- 15.7%). However, no statistical differences were observed in either the percentage of transferable embryos or in the percentage of embryos actually transferred or frozen in the two groups. In conclusion, spermatozoa from ejaculates that are washed out from the seminal fluid without any further selection can be used for ICSI without any adverse effect on fertilization and embryo cleavage.      

Effect of cetirizine on histamine- and leukotriene D4-induced bronchoconstriction in patients with atopic asthma.

Cetirizine, a derivative of hydroxyzine, is a new compound with potent antihistaminic property without antiserotonin and anticholinergic activities. The effect of both a single dose (15 mg) and 7 days of treatment (15 mg twice daily) with cetirizine, a potent H1 antagonist on bronchoconstriction induced by histamine and leukotriene D4 (LTD4) has been examined in 10 patients with mild atopic asthma in a placebo-controlled, double-blind, crossover study. Cetirizine, after a single dose and 7 days of treatment with placebo, the geometric mean values of the provocative concentration of histamine causing a 20% fall in FEV1 (millimolars) were 1.60 (95% confidence interval, 0.82 to 3.11) and 1.67 (0.77 to 3.65), compared with 118.07 (77.22 to 180.54) (p less than 0.0001) and 53.16 (20.50 to 137.84) after cetirizine administration (p less than 0.0002). The mean inhibition after a single dose was twofold higher than after 1 week of treatment (p less than 0.05). After a single dose and 7 days of treatment with placebo, the geometric mean values of the provocative concentration of LTD4 causing a 20% fall in FEV1 (micromolars) were 2.26 (1.74 to 2.94) and 2.37 (1.77 to 3.17), compared with 3.90 (2.60 to 5.86) (p less than 0.05) and 3.21 (2.28 to 4.52) after cetirizine administration. This result suggests that cetirizine is a potent H1 antagonist in the human airways. Diminished activity after 1 week of treatment suggests subsensitivity of H1 receptors developing in human airways. The small protective effect after a single dose against LTD4-induced bronchoconstriction indicates a nonspecific rather than a specific receptor antagonism.     

Accuracy of identification and susceptibility results by direct inoculation of Vitek 2 cards from positive BACTEC cultures

Rapid identification of bacteria and prompt acquisition of susceptibility results are valuable for patient care. The objective of the present study was to determine the accuracy of direct inoculation of Vitek 2 cards from positive BACTEC cultures compared to inoculation of the cards from subculture plates. Positive BACTEC cultures sampled between March 2001 and June 2002 were included. The results of direct inoculation were compared with the results of inoculation of Vitek 2 cards from subcultures. Of 161 gram-negative bacilli, 129 (80%) were correctly identified by direct inoculation compared to 145 of 161 (90%) by subculture. Susceptibility testing was performed on 2,862 antibiotic-isolate combinations. The essential agreement was 98.7%. The number of very major, major, and minor errors was 1 (0.2% of resistant strains), 1 (0.04% of susceptible strains), and 68, respectively. Direct identification of Staphylococcus spp. was not performed, but antimicrobial susceptibility was tested using 6,042 antibiotic-isolate combinations. The essential agreement was 95.2%. The number of very major, major, and minor errors was 73 (4.5% of resistant strains), 32 (0.8% of susceptible strains), and 106, respectively. Eighty-four percent of the very major errors occurred with trimethoprim-sulfamethoxazole. The results show that direct inoculation of Vitek cards is valuable as a rapid routine method for identification and susceptibility testing of gram-negative bacilli. For Staphylococcus spp., the susceptibility results obtained after direct inoculation of Vitek 2 cards are also acceptable except for those obtained with trimethoprim-sulfamethoxazole. Susceptibility results for this antibiotic, if obtained using direct inoculation, should not be reported to the clinician.     

Reproductive planning after genetic counselling: a perspective from the last decade

Studies from the last decade on factors influencing reproductive planning after genetic counselling were reviewed. Increased possibilities of DNA-analysis and prenatal diagnosis might have brought about a shift in the paramountcy of factors influencing reproductive planning after genetic counselling. In contrast to the literature in the seventies, the magnitude of the genetic risk was no longer found to be one of the decisive factors in postcounselling reproductive planning. Instead, the interpretation of the risk as high or low and the desire to have children appeared to be paramount. The impact of new developments in DNA-analysis in prenatal diagnosis and presymptomatic testing will be an important subject for future studies on factors influencing reproductive planning.     

Enhanced Immunological Memory Responses to Listeria monocytogenes in Rodents, as Measured by Delayed-Type Hypersensitivity (DTH), Adoptive Transfer of DTH, and Protective Immunity, following Lactobacillus casei Shirota Ingestion

We have investigated the effect of orally administered Lactobacillus casei Shirota (L. casei) on immunological memory, as measured by delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR). The studies were performed in animal models in which the animals were rendered immune by a primary Listeria monocytogenes infection. It was shown that orally administered viable L. casei, and not heat-killed L. casei, enhanced significantly the antigen-specific DTH at 24 and 48 h in Wistar rats, Brown Norway rats, and BALB/c mice in a time- and dose-dependent fashion. L. casei had to be administered at least 3 days prior to the DTH assay at a daily dose of 10⁹ CFU in order to induce significant effects. Long-term administration of 10⁹ CFU of viable L. casei resulted in enhanced ACR, as demonstrated by reduced L. monocytogenes counts in the spleen and liver and diminished serum alanine aminotransferase activity after reinfection. Enhancement of cell-mediated immunological immune responses by L. casei was further established in an adoptive transfer study. Naïve recipient BALB/c mice, which were infused with nonadherent, immunized spleen cells from L. casei-fed donor BALB/c mice, showed significantly enhanced DTH responses at 24 and 48 h compared to recipient mice which received spleen cells from control donor mice. In conclusion, orally administered L. casei enhanced cell-mediated immunological memory responses. The effects relied on lactobacillus dose and viability as well as timing of supplementation and, further, appeared to be independent of host species or genetic background.     

Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmic reticulum damage

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.     

Chromosomal radiosensitivity of breast cancer with a CHEK2 mutation

Recently, multiple studies have shown that a sequence variant in CHEK2 (CHEK2 1100delC) plays a role in the susceptibility to breast cancer. This mutation should confer about a twofold increased breast cancer risk in women and a 10-fold increased risk in men. Because the CHEK2 gene plays a critical role in DNA damage repair and the CHEK2 1100delC variant confers susceptibility to breast cancer, we investigated if patients carrying the CHEK2 1100delC mutation are characterized by an enhanced chromosomal radiosensitivity. To this end, familial breast cancer patients, sporadic breast cancer patients, and healthy women, considered in our previously studied to determine their chromosomal radiosensitivity with the G2 and G0-MN assay, were all tested in present study for the presence of the CHEK2 1100delC variant. The 1100delC variant was detected in none of the 100 healthy individuals, in 1 of 100 (1%) unselected breast cancer patients and in 3 of 78 (3.8%) breast cancer patients with a family history of breast cancer. The breast cancer patients with the CHEK2 1100delC genotype had a mean radiation-induced yield of chromatid breaks that was not significantly different from that of the healthy control group. Although the mean yield of micronuclei (MN) was significantly higher compared to the healthy control group, this higher mean MN yield was due to a single patient who had a very high number of MN compared to the parallel control. Our data suggest that breast cancer patients with a CHEK2 1100delC mutation are in general not characterized by a distinct enhanced chromosomal radiosensitivity. These conclusions are, however, very preliminary, because of the small numbers of CHEK2 1100delC breast cancer patients studied.     

Encoding of visceral noxious stimuli in the discharge patterns of visceral afferent fibres from the colon

Afferent fibres, in the inferior splanchnic nerves and lumbar white rami, which supply the colon and its mesentery in the cat, were investigated for their responses to distension and contraction of the colon and to local pressure applied to colon and its mesentery. 1) 63% (177 out of 287) of the axons had resting activity (median 0.3 imp/s). These axons were either unmyelinated (conduction velocity below 2 m/s) or thin myelinated (conduction velocity below 18 m/s). Most axons without resting activity (N=95 out of 106 axons) conducted at less than 1.4 m/s, and most were probably sympathetic efferents. 2) 76 out of 80 afferent units with resting activity (95%) and 8 out of 27 units without (30%) were excited by distension of the colon. The thresholds were largely at intraluminal pressures of around 25 mm Hg or higher. 3) Most afferent units (87%) responded with an increased steady state discharge throughout the distension with or without initial dynamic response. The rest of the afferent units responded only with a transient discharged to distension. 4) Most afferent units reacted in a graded manner to variable intraluminal pressures. 5) In only 43% of the distension-sensitive afferent units could mechanoreceptive sites be located on the wall of the colon or in the mesentery. The majority of the afferent units had one mechanoreceptive site only, some had two. 6) Afferent units reacting to colon distension were also excited by contraction of the colon. 7) The excitability spectrum of visceral afferent fibres in the inferior splanchnic nerves, which are activated by colon distension, suggests that these units are involved in visceral nociception from the colon.