Emergence of resistance to beta-lactam agents inPseudomonas aeruginosa with group I beta-lactamases in Spain

The contribution of induction and stable derepression of chromosomal class I -lactamases to -lactam antibiotic resistance was studied in clinical isolates ofPseudomonas aeruginosa collected from patients treated with -lactam antibiotics. Multiple isolates from the same patient were characterized by O-serotyping as a primary screen, combined with pyocin typing. Sonicated extracts of cells were assayed for chromosomal and plasmid-mediated -lactamases by isoelectric focusing and cloxacillin inhibition studies. The specific -lactamase activity, basal and induced, with cefoxitin was determined to differentiate strains with inducible or derepressed production of the enzyme. Beta-lactamase induction was performed in each strain against the -lactam agents used in the therapy of each patient. The observations showed that induction against older penicillins such as penicillin, amoxicillin, and amoxicillin/clavulanate resulted in a moderate to strong increase in -lactamase activity, whereas the results obtained with first-generation cephalosporins varied with the -lactam agent tested. Third-generation cephalosporins were weak inducers of -lactamases, and their use as therapy preceded the appearance of strains that produce chromosomal group I -lactamases constitutively. These strains showed a remarkable reduction in sensitivity to ureidopenicillins, carboxipenicillins, third-generation cephalosporins, and monobactams, but not to carbapenems.     

Activity of beta-lactamase inhibitor combinations onEscherichia coli isolates exhibiting various patterns of resistance to beta-lactam agents

The efficacy of the clinically available -lactam/-lactamase inhibitor combinations (amoxicillin/clavulanic acid (CA), ticarcillin/CA, amoxicillin/sulbactam, and piperacillin/ tazobactam) was evaluated on 300 amoxicillin-resistantEscherichia coli isolates having the main patterns of -lactam resistance. The patterns, which reflect the production of various -lactamase enzymes, were analyzed by a principal component analysis of susceptibility to 11 -lactam antibiotics or -lactam/-lactamase inhibitor combinations. Sixty-two percent of strains were not very susceptible to penicillins, cephalothin, or any -lactam/-lactamase inhibitor combinations except for piperacillin/tazobactam; these strains may represent high-level broad-spectrum -lactamase (so-called penicillinase) production phenotype or inhibitor-resistant TEM-like enzyme production phenotype. Of the strains, 14.7 % were resistant to amoxicillin and ticarcillin compatible with low-level broad-spectrum -lactamase production phenotype; 5.7 % were cefoxitin resistant and were postulated to present a high-level cephalosporinase production phenotype; and 2.6 % were resistant to cephalothin only, attributable to a low-level cephalosporinase production phenotype. Three percent of strains were intermediate or resistant to cefotaxime and may produce an extended-spectrum -lactamase, and the remaining strains (12 %), resistant to all tested antibiotics except for cefotaxime and piperacillin/tazobactam, were hypothesized to produce both broad-spectrum -lactamase plus cephalosporinase. The minimal inhibitory concentration (MIC) for these phenotype patterns indicated that combinations of CA plus amoxicillin or ticarcillin, or sulbactam plus amoxicillin, restored the activity of penicillins against phenotype 1 strains, whereas these combinations remained inactive against the other phenotype strains. Piperacillin plus tazobactam showed the best in vitro effect against the strains of all resistance phenotypes.     

In vitro activity of biapenem against beta-lactamase producingEnterobacteriaceae

The activity of biapenem was compared with that of imipenem and cefotaxime against 108 strains of -lactamase producingEnterobacteriaceae. Biapenem and imipenem were very active, inhibiting 90 % of the strains at a concentration of 0.5 µg/ml. Both carbapenems were very active against plasmidic -lactamase producers, with MIC90s below 1 µg/ml. However, the MIC90 of biapenem for cephalosporinase producers was 1 µg/ml. Against strains producing extended-spectrum -lactamases, biapenem exhibited better activity against TEM-type producers (MIC90 0.25 µg/ml) than against SHV-type producers (MIC90 0.5 µg/ml). Overall, the in vitro antibacterial activity of biapenem is similar to that of imipenem.     

Dissemination of cephalosporin-resistantSerratia marcescens strains producing a plasmidic SHV type beta-lactamase in Greek hospitals

The resistance to third generation cephalosporins in nineSerratia marcescens strains isolated in Greek hospitals was studied. Eight of the strains transferred resistance toEscherichia coli by means of large plasmids that encoded for an extended-spectrum -lactamase. Hybridization, isoelectric focusing and hydrolysis studies showed that the enzyme resembled the SHV-5 -lactamase. In the eight isolates that possessed the SHV type enzyme, cephalosporinase expression was inducible, whereas the remaining strain was a cephalosporinase hyperproducing strain. Introduction of a plasmid coding for the regulatoryampD gene in the latter strain eliminated -lactamase production and rendered the strain susceptible to cephalosporins.     

In vivo acquisition of extended-spectrum beta-lactamase inSalmonella enteritidis during antimicrobial therapy

The recovery of aSalmonella enteritidis strain that acquired resistance to -lactams (including cefotaxime), to aminoglycosides and to chloramphenicol subsequent to cefotaxime therapy is reported. This resistance pattern to -lactams was due to the presence of an extended-spectrum -lactamase. The isoelectric point of this extended-spectrum -lactamase was 6.3. The resistance genes were located on a transferable high-molecular-weight plasmid.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Participation of β-endorphin in the regulation of conditioned reflex activity of cats

The role of -endorphin in the regulation of conditioned instrumental food-procuring reactions and more complex forms of nervous activity such as reflexes of choice of the side of reinforcement was studied in cats. It was established that the subcutaneous injection of small doses of -endorphin (10 g/kg, 15 ·10^–6 g/kg) exerts a facilitatory nonspecialized effect on positive and negative food-procuring conditioned reflexes that has an overall adaptive character overall. The influence of the same doses of -endorphin on conditioned reflexes of choice is more complex in character, depending upon the initial level of conditioned reflex activity and on the typological features of the experimental animals. A possible mechanism of the influence of -endorphin on higher nervous activity is discUSSed.Deceased.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 76, No. 8, pp. 992–1000, August, 1990.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

Occurrence and regional distribution of SHV-type extended-spectrum β-lactamases in Hungary

In order to determine the presence and geographical distribution of SHV-type extended-spectrum -lactamase genes among Enterobacteriaceae strains in Hungary, isolates from 25 microbiology laboratories throughout the country were collected between January 2002 and August 2003 and examined. Sequencing of the genes showed that SHV-5 and SHV-2a are the dominant SHV-types in extended-spectrum -lactamase-producing Enterobacteriaceae strains in this country. The SHV-2 gene, which is prevalent in many European countries, was not detected, but one isolate carried the SHV-12 gene. The results show that these genes are circulating among Enterobacteriaceae strains in Hungary and indicate that strict infection control measures are warranted in order to prevent their spread.     

Assessment of plasma opioid peptides, β-endorphin and met-enkephalin,at the end of an international nordic ski race

Summary Plasma met-enkephalin, -endorphin, cortisol and lactic acid concentrations were measured in seventeen volunteer male subjects at rest and after a long-distance nordic ski race. Immediately after the race, mean plasma met-enkephalin did not show any significant change, but significant rises in -endorphin, cortisol and lactic acid were noted in all skiers. The change in -endorphin with exercise was significantly related to the change in cortisol (r=0.68;p<0.001) and to the change in plasma lactic acid (r=0.60;p<0.001). Furthermore, the experienced skiers training over 150 km · week^–1 of nordic ski had significantly faster skiing times in this event and showed greater -endorphin, cortisol and lactic acid levels than the recreational skiers who trained for 20 km · week^–1. Our results imply that the changes in plasma -endorphin depend on the intensity of exercise. However the significance of higher levels of skiing training or previous nordic ski experience in the release of -endorphin is expected and cannot be excluded.     

Susceptibility ofAlcaligenes denitrificans subspeciesxylos-oxydans to beta-lactam antibiotics

The susceptibility of 56 clinical isolates and two reference strains ofAlcaligenes denitrificans subsp.xylosoxydans to -lactam agents was tested and related to -lactamase activity of the strains. The MICs of 12 -lactams determined by an agar dilution method showed that all the strains were sensitive to imipenem and moxalactam. Forty-one cloxacillin-sensitive -lactamase producing strains were highly susceptible to azlocillin, piperacillin and ticarcillin, and less susceptible to several cephalosporins (cefamandole, cefoperazone, ceftazidime). The 17 remaining -lactamase-producing strains, which were sensitive to clavulanic acid and to a lesser extent cloxacillin, had variable resistance to the penicillins tested and synergy was obtained when these penicillins were combined with clavulanic acid or tazobactam. The choice of agents for treatment of infections with this organism must take into account the susceptibility phenotype of clinical isolates.     

Interleukin-1β induces expression of cyclooxygenase-2 mRNA in human gingival fibroblasts

The effect of interleukin-1 (IL-1) on the expression of cyclooxygenase-1 and –2 (COX-1 and COX-2) mRNA and its relation to prostaglandin E₂ (PGE₂) biosynthesis in human gingival fibroblasts was studied. IL-1 increased levels of mRNA for COX-2 whereas the COX-1 mRNA level was unaffected. The increased COX-2 mRNA levels were accompanied by enhanced PGE₂ formation. The phorbol, 12-myristate 13-acetate (PMA), known to stimulate protein kinase C (PKC), also induced expression of COX-2 mRNA. When gingival fibroblasts were treated simultaneously with IL-1 and PMA, the cytokine IL-1 synergistically increased levels of COX-2 mRNA, accompanied by a corresponding increase in PGE₂ biosynthesis. The anti-inflammatory steroid, dexamethasone (DEX) abolished the enhanced expression of COX-2 mRNA as well as PGE₂ formation induced by IL-1, PMA or the combination of IL-1 and PMA. The study indicates that the IL-1 induced PGE₂ formation is mediated by an enhanced gene expression of COX-2 in gingival fibroblasts suggesting that the enzyme COX-2 may play an important role in the regulation of prostanoid formation at inflammatory lesions in gingival tissue.     

Beta-lactamase production and susceptibility of US and European anaerobic gram-negative bacilli to beta-lactams and other agents

The susceptibility of 1,476 US and European strains of anaerobic gram-negative bacilli to amoxicillin, amoxicillin/clavulanate, ticarcillin, ticarcillin/clavulanate, cefoxitin, imipenem and metronidazole was determined. All of theBacteroides fragilis group and 51 % of the non-Bacteroides fragilis group were -lactamase positive. Amongst the non-Bacteroides fragilis group, -lactamase positivity rates were higher for US strains (58 %) than for European strains (39 %). All strains were susceptible to imipenem and metronidazole. MIC90s of amoxicillin and ticarcillin for all -lactamase negative strains were 0.5 and 2 µg/ml, respectively. The addition of clavulanate reduced the MIC90s of amoxicillin ( 256 µg/ml) and ticarcillin ( 64 µg/ml) to 16 and 8 µg/ml, respectively, for theBacteroides fragilis group, and to 4 µg/ml for both agents for the non-Bacteroides fragilis -lactamase producing group. Twenty-nine cefoxitin-resistant strains were found, mainly in theBacteroides fragilis group, while 95 -lactamase producing strains (predominantlyBacteroides fragilis group and fusobacteria) did not show synergy between -lactams and clavulanate. Of the newer agents tested, meropenem and piperacillin-tazobactam were the most active (100 % of strains susceptible), followed by amoxicillin-BRL 42715 (99 % of strains susceptible); 94 to 98 % of the strains were susceptible to cefoperazone-sulbactam, tosufloxacin, tempafloxacin and clindamycin. Only 73 % of the strains were susceptible to cefotetan, compared to 91 % to cefoxitin; 88 % of the strains were susceptible to trospectomycin. Overall, all of the -lactam/-lactamase inhibitor combinations, imipenem, meropenem, cefoxitin, tosufloxacin, temafloxacin and clindamycin had good activity against -lactamase producing strains, while all agents tested had good activity against -lactamase negative strains.     

Lack of additive effect between mechanisms of resistance to carbapenems and other beta-lactam agents inPseudomonas aeruginosa

Eighty-nine clinical isolates resistant (n=61) or susceptible (n=28) to imipenem and exhibiting the main patterns of susceptibility to other -lactam agents (wild type pattern, penicillinase pattern, constitutive cephalosporinase pattern) were studied in order to investigate (i) the mechanism of resistance involved and (ii) whether resistance to carbapenems affects the level of resistance to other -lactam agents and, conversely, if resistance to other -lactam agents affects the level of resistance to carbapenems. For this purpose, the presence of OprD protein in the cell wall was detected by Western blot and -lactamase activity by spectrophotometric assay and isoelectric focusing. OprD expression was not detectable in the imipenem-resistant (MIC16 g/ml) strains. It was decreased in half the strains for which MICs of imipenem were 2 to 8 g/ml and was close to a normal level in the most susceptible strains (MIC 1 g/ml), thus demonstrating a direct correlation between the level of susceptibility to imipenem and the level of OprD expression. No imipenemase activity was detected in imipenem-resistant strains. Synergy between imipenem or meropenem and BRL42715 was observed for all of the strains, demonstrating the role of cephalosporinase in carbapenem resistance. Within each pattern of susceptibility, the mean MICs of -lactam agents other than carbapenems were similar, whether the strains were susceptible or resistant to imipenem. Conversely, the mean MICs of imipenem or meropenem for either the imipenem-resistant or the imipenem-susceptible strains were similar, regardless of the susceptibility of these strains to the other -lactam agents. Thus, when several mechanisms of resistance to -lactam agents are present in the same strain ofPseudomonas aeruginosa, there is no additive effect between these mechanisms.     

Estradiol attenuation of beta-amyloid-induced toxicity: a comparison o.

17-estradiol (E2) has been shown to attenuate the toxicity of -amyloid peptides (A) in neuronal cultures with the effective concentration of E2 ranging from low nM to high M. This study compares the effective neuroprotective concentration of E2 against both A-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective E2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum E2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM E2 conferring significant protection in the calcein AM assay but 1 M E2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic A concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM E2 against H_{2 2} toxicity. These results indicate that low concentrations of E2 can attenuate A and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of E2-mediated attenuation of A toxicity.     

Interspecific genetic complementation analysis of human and sheep fibroblasts withβ-galactosidase deficiency

Interspecific somatic cell hybrids were analyzed by genetic complementation to determine if a lysosomal storage disease in sheep associated with deficiencies of -galactosidase and -neuraminidase was homologous with any of four -galactosidase-deficient human diseases. Fibroblasts from -glactosidase-deficient sheep, cats, and human patients were fused and assayed histochemically for -galactosidase, with 5-bromo-4-chloro-3-indolyl -d-galactoside. We observed complementation in heterokaryons consisting of fibroblasts from -galactosidase-deficient sheep and fibroblasts from patients with galactosialidosis or mucolipidosis type II, but no complementation in heterokaryons consisting of fibroblasts from -galactosidase-deficient sheep and fibroblasts from human or feline GM₁, gangliosidosis (type I) or from human mucopolysaccharidosis type IVB fibroblasts. We conclude that the ovine disease is due to a mutation at the genetic locus homologous with that of GM₁, gangliosidosis and mucopolysaccharidosis type IVB, suggesting that the primary defect in the ovine disease is a mutation of the -galactosidase structural gene.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Localization of Triton-insoluble cAMP-dependent kinase type RIbeta in rat and mouse brain

In eukaryotic cells, cAMP regulates many different cellular functions. Its effects are in most cases mediated by cAMP-dependent protein kinases. These consist of two regulatory and two catalytic subunits. In mammals, four different isoforms of cAMP-dependent protein kinases regulatory subunits have been characterized (RI and , RII and ). These four isoforms show a high level of homology and slightly different biochemical properties. In addition to biochemical properties, a different anatomical distribution of the regulatory isoforms may contribute to determine the specificity of diverse cAMP effects. By immunohistochemistry, the distribution of the detergent-insoluble fraction of RI isoform has been examined in rat and mouse brain. Biochemical fractionation shows that a large fraction of both RI and RI isoforms is bound to the cytoskeleton. RI labelling can be observed only in few locations: Purkinje cells, olfactory mitral cells, lateral thalamic neurons, superior olivary complex neurons. These cell populations are involved in the so called Purkinje cell degeneration. On the other hand, RI aggregates have a more widespread distribution, in brain areas involved in visceroemotional control. At the subcellular level, these two subunits show a different pattern of labelling: in most cells a sharply defined clustered labelling is observed for RI isoforms, while the RI isoform presents a weaker, diffuse intracytoplasmic distribution. Competition experiments point to the presence of, as yet unidentified, different and selective anchoring proteins for the two similar RI and isoforms. It is suggested that, as is the case for structural proteins, a different supramolecular organization of similar regulatory proteins may be crucial in order to fulfill different functions.     

Mapping thyrotropin β subunit gene in man and mouse

Thyrotropin (TSH) is composed of two subunits: and . Previously, we have mapped the TSH gene to human chromosome 6 and mouse chromosome 4. In this study we have located the human TSH gene on chromosome 1 and the mouse TSH gene to chromosome 3. These data suggest that the TSH gene lies in a conserved linkage group with the genes for amylase 1 and 2, nerve growth factor, and the protooncogene Nras.