Evaluation of PCR-Based Testing for Surveillance of KPC-Producing Carbapenem-Resistant Members of the Enterobacteriaceae Family

The spread of carbapenem-resistant members of the Enterobacteriaceae family (CRE) harboring carbapenemases is an emerging public health threat. As KPC-producing Klebsiella species are endemic in our tertiary care hospital, we aimed to evaluate a PCR-based surveillance test for identification of rectal carriage of KPC-producing CRE. We conducted a surveillance study between May and December 2007. Rectal swabs were collected from patients known to harbor CRE and from contacts of newly discovered patients harboring CRE. Specimens were evaluated by culture and by PCR analysis for bla_KPC and were defined as positive if CRE was cultured and bla_KPC was identified. Discrepant results between the culture and PCR analysis were resolved by subculturing, repeating the PCR, and performing a hydrolysis assay. Positive CRE cultures prior or subsequent to the time of sampling for the study were also taken into consideration. Sensitivity, specificity, and time to result were calculated. A total of 755 swabs were included. Concordant results were documented for 735 specimens; 51 were positive as determined by both PCR and culture. Discrepancies existed for 20 swabs; 9 were bla_KPC negative and CRE culture positive, and 11 were bla_KPC positive and CRE culture negative. After repeat testing, a total of 64 samples were classified as bla_KPC-positive CRE. The sensitivity and specificity of the PCR analysis were 92.2% and 99.6%, respectively, and those of the culture were 87.5% and 99.4%, respectively. Over the last 3 months of the study, the sensitivity of the PCR improved to 96.3%, versus 77.8% for culture. Time to result was 30 h for the PCR and 60 h (negative) and 75 h (positive) for the CRE culture. bla_KPC PCR-based testing is a useful method for the surveillance of KPC-producing CRE. Its main advantage over culturing is a shorter time to result, and it may prove to be more sensitive.KPC-mediated carbapenem resistance in members of the Enterobacteriaceae has emerged recently in Israel, as observed in clinical strains of Escherichia coli (14), Enterobacter cloacae (13), and Klebsiella pneumoniae (10). An epidemic clone of KPC-3-producing Klebsiella pneumoniae, possessing resistance to nearly all antimicrobial agents, excluding gentamicin and colistin, has spread in all major Israeli hospitals (15). Infections caused by this strain have been associated with greater than 40% in-hospital mortality (23). As part of our extensive infection control efforts to limit the spread of this highly epidemic strain in the hospital, we aimed to implement a policy of early detection of carriers by screening for gastrointestinal carriage of this pathogen.Active surveillance has been shown to be an effective infection control strategy with other antibiotic-resistant pathogens, such as methicillin (meticillin)-resistant Staphylococcus aureus (4, 5) and vancomycin-resistant enterococci (16). The use of culture-based methods for detection of carbapenem-resistant members of the Enterobacteriaceae family (CRE) from rectal or stool surveillance samples was evaluated previously, using selective and nonselective broth media supplemented with an imipenem disk, followed by plating on MacConkey agar with and without imipenem (9). PCR-based assays were also assessed for detection of carbapenem-resistant K. pneumoniae from rectal surveillance swabs (6, 8). Although good performance was shown by both methods, there is still no recommended reference method for the recovery of CRE in the context of active surveillance. Moreover, automated susceptibility testing systems may fail to detect KPC-mediated resistance in clinical isolates (1, 24).We aimed to implement a rapid test for identification of CRE gastrointestinal carriage based on a molecular method. Since in our institution carbapenem resistance in members of the Enterobacteriaceae, and specifically in the epidemic K. pneumoniae strain, is rendered by the presence of bla_KPC, we designed and evaluated a PCR-based method for bla_KPC detection in rectal specimens. We compared the performance of this newly developed molecular-based diagnostic tool with that of a culture-based method for identifying carriers of KPC-producing CRE.(This work was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy/46th Annual Meeting of the Infectious Diseases Society of America, Washington, DC, 25 to 28 October 2008 [20].)     

Extended-spectrum beta-lactamase production is associated with an increase in cell invasion and expression of fimbrial adhesins in Klebsiella pneumoniae

Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strains are suggested to possess higher pathogenic potential than non-ESBL producers. Microbial adherence to and invasion of host cells are critical steps in the infection process, so we examined the expression of type 1 and 3 fimbrial adhesins by 58 ESBL-producing and 152 nonproducing isolates of K. pneumoniae and their abilities to invade ileocecal and bladder epithelial cells. Mannose-sensitive hemagglutination of guinea pig erythrocytes and mannose-resistant hemagglutination of ox erythrocytes were evaluated to determine the strains’ abilities to express type 1 and type 3 fimbriae, respectively. Bacterial adhesion to and invasion of epithelial cells were tested by enzyme-linked immunosorbent assay and imipenem killing assay, respectively. The adherence of ESBL- and non-ESBL-producing strains to epithelial cells did not differ significantly (P > 0.05). In contrast, the proportion of strains capable of invading (>5% relative invasion) ileocecal and bladder epithelial cells was significantly higher among ESBL producers (81%, n = 47/58, and 27.6%, n = 16/58, respectively) than among non-ESBL producers (61%, n = 93/152, and 10%, n = 15/152, respectively) (P = 0.0084, odds ratio [OR] = 2.711, 95% confidence interval [CI] = 1.302 to 5.643 and P = 0.0021, OR = 4.79, 95% CI = 1.587 to 7.627). The mean invasion by ESBL producers (5.5% ± 2.8% and 3.3% ± 2.7%, respectively) was significantly higher than that by non-ESBL producers (2.9% ± 2.6% and 1.8% ± 2%, respectively) (P < 0.0001). Likewise, the proportion of ESBL producers coexpressing both fimbrial adhesins was significantly higher (79.3%; n = 46/58) than that of non-ESBL producers (61.8%; n = 94/152) (P = 0.0214; OR = 2,365; 95% CI = 1.157 to 4.834). Upon acquisition of SHV-12-encoding plasmids, two transconjugants switched on to produce type 3 fimbriae while expression of type 1 fimbriae was not affected. The acquisition of an ESBL plasmid appeared to upregulate the phenotypic expression of one or more genes, resulting in greater invasion ability.     

Hand dominance in upper extremity musculoskeletal disorders

OBJECTIVE: To investigate the role of hand dominance in common upper extremity musculoskeletal disorders (UEMSD) in a population study. METHODS: The target population consisted of a representative sample of people aged 30 years or older residing in Finland during 2000-2001. Of the 7977 eligible subjects, 6254 (78.4%) were included in the study. RESULTS: The prevalence of UEMSD was as follows: rotator cuff tendinitis 3.8%, bicipital tendinitis 0.5%, lateral epicondylitis 1.1%, medial epicondylitis 0.3%, carpal tunnel syndrome (CTS) 3.8%, and surgery due to CTS 1.3%. CTS was 2.5 times as prevalent in women as men, whereas the other UEMSD were as common in both sexes. Rotator cuff and bicipital tendinitis and medial epicondylitis were more prevalent in the dominant arm only in women, whereas lateral epicondylitis was more prevalent in the dominant elbow in both sexes. The higher prevalence of rotator cuff and bicipital tendinitis in the dominant side persisted beyond working age. The prevalence of CTS did not differ by hand dominance. Dominant hand had been operated more frequently for CTS in women. CONCLUSION: Our findings show that UEMSD are more prevalent in the dominant than nondominant arm mainly in women. For shoulder tendinitis, the difference persists throughout adult age. Physical load factors may have long-lasting effects on the shoulder and they may play a greater role in women than men.     

Vitamin D and diabetes mellitus

Better understanding of the physiological role of the vitamin-D system, in particular its potential effects on inflammatory and autoimmune conditions as well as on insulin secretion and possibly also on insulin resistance, increased the interest in its potential role in prevention and control of the diabetic condition, both type-1 and -2 diabetes. Both these conditions are associated with inflammation and type-1 diabetes also with an autoimmune pathology. Indeed, animal and human studies support the notion that adequate vitamin-D supplementation may decrease the incidence of type-1 and possibly also of type-2 diabetes mellitus and may improve the metabolic control in the diabetes state. However, the exact mechanisms by which vitamin-D and calcium supplementation exert their beneficial effects are not clear and need further investigation.     

Changes of spine density and dendritic complexity in the prefrontal cortex in offspring of mothers exposed to stress during pregnancy

Both chronic stress in adulthood and episodes of stress in the early postnatal period have been shown to interfere with neuronal development in limbic prefrontal cortical regions. The present study in rats showed for the first time that the development of layer II/III pyramidal neurons in the dorsal anterior cingulate (ACd) and orbitofrontal cortex (OFC) is significantly affected in offspring of mothers exposed to stress during pregnancy. In prenatally stressed (PS) male rat pups the ACd and OFC showed significantly lower spine densities on the apical dendrite (ACd, -20%; OFC, -25%), on basal dendrites reduced spine densities where found only in the OFC (-20% in PS males). Moreover, in both cortical areas a significant reduction of dendritic length was observed in PS males compared to control offspring, which was confined to the apical dendrites (ACd, -30%, OFC, -26%). Sholl analysis revealed that these alterations were accompanied by a significantly reduced complexity of the dendritic trees in both cortical regions. PS females displayed reductions of dendritic spine densities in the ACd and OFC on both the basal (ACd, -21%; OFC, -20%) and apical dendrites (ACd, -21%; OFC, -21%), however, in contrast to the findings in PS males, no dendritic atrophy was detected in the PS females. These findings demonstrate that gestational stress leads to significant alterations of prefrontal neuronal structure in the offspring of the stressed mothers in a sex-specific manner.     

Detection of differentially expressed glycogenes in trabecular meshwork of eyes with primary open-angle glaucoma

PURPOSE: To identify differentially expressed glycogenes in trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG). METHODS: Total RNA was isolated from TM of cadaveric eyes derived from donors with diagnosed glaucomas of different etiologies and from normal control subjects. RNA was amplified and hybridized to the GLYCOv2 oligonucleotide microarray that contains probes for carbohydrate-binding proteins, glycosyltransferases, and other genes involved in the regulation of glycosylation. Statistical analysis was used to identify differentially expressed genes between normal and POAG samples. RESULTS: This study revealed that POAG TM and normal TM have distinct gene expression profiles. Of the 2001 genes on the array, 19 genes showed differential expression of greater than 1.4-fold in POAG. Mimecan and activinA, which have been shown to be upregulated in models of glaucoma, were both found to be elevated in POAG TM. Many genes were identified for the first time to be differentially regulated in POAG. Among the upregulated genes were: (1) cell adhesion molecules including platelet endothelial cell adhesion molecule-1 and P-selectin, both of which are targets of NFkappaB, which has been shown to be activated in glaucomatous TM; (2) lumican, a core protein of keratan sulfate proteoglycans; and (3) the receptor for IL6, a cytokine that has been shown to be upregulated in TM in response to elevated intraocular pressure. Among the downregulated genes were chondroitin-4-O-sulfotransferase involved in the synthesis of chondroitin sulfate chains and the receptor for PDGFbeta, a growth factor that has been shown to stimulate both TM cell proliferation and phagocytic activity. Results for several genes were confirmed by RTq-PCR. CONCLUSIONS: Microarray technology was used to show, for the first time, that POAG TM has a distinct glycogene expression profile. Differentially expressed glycogenes identified in this study have not been previously investigated for their role in the pathogenesis of POAG and thus are novel factors for further study of the mechanism of the disease and for their possible use as diagnostic markers.     

Pandangolide 1a, a metabolite of the sponge-associated fungus Cladosporiumsp., and the absolute stereochemistry of pandangolide 1 and iso-cladospolide B

Fractionation of the ethyl acetate extract of a Cladosporium sp., isolated from the Red Sea sponge Niphates rowi, yielded a new hexaketide, pandangolide 1a (1), together with its known diastereomer pandangolide 1 (2) and the known iso-cladospolide B (3). The structures of the compounds were determined by 1D and 2D NMR techniques and mass spectrometric data. The absolute configurations of the stereocenters in these compounds were determined by Riguera's method and circular dichroism.     

Protection against ischemia/reperfusion injury by the cavitary two-layer method in canine small intestinal transplantation with reduction of reactive oxygen species

BACKGROUND: Ischemia and reperfusion (I/R) injury is a major determinant of early graft dysfunction and long-term graft survival in small intestinal transplantation. The cavitary two-layer method (TLM) has been reported to be superior to the University of Wisconsin cold storage method (UWM) in long-term preservation of canine small intestine. This study was designed to evaluate the protective effect of the cavitary TLM against I/R injury in canine small intestinal transplantation. METHODS: Intestinal grafts harvested from beagles were allotransplanted after 24-hour preservation by UWM (group 1) or the cavitary TLM (group 2). The graft in the controls (group 3) was immediately allotransplanted without preservation. I/R injury was assessed by functional success rates, biochemical assay, graft adenosine triphosphate (ATP) and lipid peroxidation (LPO) concentrations, and histopathologic examination including TUNEL staining for apoptosis. RESULTS: In group 1, ATP recovery was delayed after reperfusion, and most recipients died with hemorrhage of the grafts and lungs. In group 2, graft ATP concentrations recovered rapidly, and I/R injury was prevented with reduced LPO production, resulting in good outcome. CONCLUSIONS: The cavitary TLM protected intestinal grafts against I/R injury evidenced by maintenance of graft ATP levels and reduction of LPO production compared with UWM in canine small intestinal transplantation.