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.     

Isolation ofEnterobacter aerogenes susceptible to beta-lactam antibiotics despite high level beta-lactamase production

This report describes a patient with nosocomial meningitis from whom four distinct isolates ofEnterobacter aerogenes were recovered over a complicated course of chemotherapy. The initial isolate was susceptible to expanded spectrum -lactams despite constitutive production of high levels of -lactamase. Resistant isolates recovered during antibiotic therapy had lost a 42,000 outer membrane protein. These data suggest that b-lactam susceptibility in the original isolate was due to hyperpermeability mediated by the 42,000 Dalton protein.     

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.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Survey of clinical isolates ofStaphylococcus aureus for borderline susceptibility to antistaphylococcal penicillins

On the basis of the MICs of methicillin and oxacillin, 975 clinical isolates ofStaphylococcus aureus were categorized as having resistance, borderline susceptibility or full susceptibility to penicillinase-resistant penicillins (PRPs). The borderline phenotype accounted for 122 isolates (12.5 %), whereas 562 isolates were fully susceptible and 290 resistant; one remaining isolate had resistance to methicillin and borderline susceptibility to oxacillin. Reductions in the MICs of methicillin and oxacillin in the presence of sulbactam were greater in strains with borderline PRP susceptibility than in fully susceptible or resistant isolates. Over 99 % of fully PRP-susceptible strains, 93 % with borderline susceptibility and 71 % of resistant strains were susceptible to ampicillin/sulbactam. The production of -lactamase, assayed in all strains using nitrocefin as substrate, could be detected without prior induction in 729 strains and after induction only in another 156 strains. With only two exceptions, the -lactamase negative strains were part of the fully PRP-susceptible group of organisms (88 of 562 isolates). Among the borderline isolates, strong -lactamase reactions were encountered with particular frequency, but not in all strains and not exclusively in borderline strains. Although associated with the majority of borderline strains, -lactamase hyperproduction thus did not appear to be an essential feature of the borderline phenotype. The results obtained may have implications for laboratory and clinical medicine, also in the light of recent findings suggesting that other mechanisms besides -lactamase hyperproduction may account for borderline susceptibility to PRPs.     

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     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

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.     

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.     

Inhibition of lung colonization of mouse colon 26 adenocarcinoma by recombinant mouse interferon β through a modification of platelet function

Recombinant murine interferon (MuIFN-) given i.v. efficiently inhibited both pulmonary arrest and formation of lung colonies of NL-17, a highly metastatic variant of mouse colon adenocarcinoma 26. NL-17 was rather resistant to MuIFN- in vitro and was highly resistant to natural killer cells of mice even though they were treated in vivo with MuIFN-. Platelets isolated from MuIFN--treated mice showed reduced aggregating activity induced by NL-17. Since lung colonization by NL-17 is influenced by platelet aggregation, the inhibition of colonization by MuIFN- could be partly mediated through modification of platelet function in vivo. The effect of MuIFN- on platelet function and its subsequent inhibition of lung colony formation give new insights into the action of recombinant MuIFN-.     

Dissemination in five French hospitals ofKlebsiella pneumoniae serotype K25 harbouring a new transferable enzymatic resistance to third generation cephalosporins and aztreonam

A strain ofKlebsiella pneumoniae K25 resistant to newer-lactam drugs was isolated in clusters in five hospitals in the Paris area. The MICs of ceftazidime and aztreonam (128 mg/l) were higher than that of cefotaxime (16 mg/l) for the strain but when measured in the presence of clavulanic acid, they were 1 mg/l. The donor strains and derivatives produced a-lactamase with a pI of 7.75–7.8 and hydrolysing activity against a wide spectrum of-lactams similar to that of SHV-2 and SHV-3, but with significant hydrolysis of ceftazidime. This new enzyme could be designated SHV-4.     

Selection and properties ofPseudomonas aeruginosa variants resistant to beta-lactam antibiotics

The relation between basal and inducible-lactamase production and resistance to-lactam compounds was studied in five clinicalPseudomonas aemginosa isolates and their corresponding resistant variants selected in the presence of either piperacillin, ceftazidime or aztreonam. In all wild-type strains enzyme levels were barely detectable in the uninduced state and most-lactams, including sulbactam and clavulanic acid, exhibited poor induction potency. Imipenem proved to be the most potent inducer in both these strains and their resistant variants. In the variants selected by either piperacillin or ceftazidime enzyme production amounted to 1.28 units/mg protein of the cell-free supernatants following the addition of-lactams as inducers. Additionally, these variants exhibited the phenomenon of non-specific induction, i.e. the increase of enzyme production by either a complex nutrient medium or by addition of vitamins. Enzyme production in the aztreonam-resistant variants was identical to that in the wild-type strains with a single exception, where the entire derepression of-lactamase production in one of the variants took place. Derepression of the chromosomally mediated enzyme affects the susceptibility to ureidopenicillins more than that to carboxy-penicillins and cephalosporins, whereas the-lactamase-independent resistance results in increased resistance to all-lactams with the single exception of imipenem.     

Synchronous appearance of fibronectin,integrin α5β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing

The distribution of integrin 51 (51) and associated components during wound healing was investigated in the rat trachea following mechanical injury. Under anesthesia, the ventral surface of the trachea was scratched, and tissue specimens were obtained from 6 h to 3 weeks after injury and studied using light and electron microscopy and immunohistochemistry. 51, vinculin and actin in regenerating epithelial cells and extracellular fibronectin appear virtually simultaneously after injury (from 12 h to 7 days) as do 51, vinculin and -smooth muscle actin in fibroblasts and cellular fibronectin in granulation tissue (from 3 to 10 days). Immunoelectron microscopy 2 days after injury showed that 51 and vinculin were localized on the basal and lateral surfaces of regenerating epithelial cells and fibroblast surfaces, and fibronectin was localized just under the regenerating epithelial cells, around collagen fibrils and sporadically around fibroblasts. Bromodeoxyuridine labeling showed that the appearance of these components was associated with the period of cell proliferation. The appearances of fibronectin, 51, vinculin and actin in regenerating epithelial cells and fibroblasts during tracheal wound healing are well coordinated. During the initial cell migration phase, plasma fibronectin may stimulate cell migration before cellular fibronectin is produced in situ, and regenerating epithelial cells appear to begin to migrate into the wound before cell proliferation starts.     

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.     

Effects of serine protease inhibitor,tame, on IL-1β in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 (mIL-1) but did not result in release of precursor IL-1 (pIL-1). In contrast, the presence of a serine protease inhibitor, N-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 as well. These effects were initially discerned from observations that the fraction of the total IL-1 produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 synthesis, predominantly affecting 31-kDa pIL-1, and a dose-dependent inhibition of TNF- production. Parallel examination of lactate dehydrogenase (LDH) release indicated thatpIL-1 release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 in situ but are probably not mechanistically related to either maturation of the IL-1 molecule or signaling of IL-1 release. IL-1 release appears to be dependent on the amount of total IL-1 synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1.     

Distribution of skeletofusimotor axons in lumbrical muscles of the monkey

Summary The nerve supply to 25 poles of muscle spindles in the monkey was reconstructed by light microscopy of serial 1-m thick transverse sections of lumbrical muscles. Twenty of 60 motor axons that supplied the spindle poles were identified as skeletofusimotor (). Twenty-eight percent of the spindle poles were innervated by axons, in addition to axons. Every -innervated spindle pole transected an endplate zone of extrafusal muscle. Most axons coinnervated extrafusal fibers rich in mitochondria and the nuclear bag₁ or nuclear chain intrafusal fibers. All but two axons innervated one type of intrafusal fiber only. The intramuscular organization of motor system in lumbrical muscles of the monkey was similar to that of the cat tenuissimus muscle. The function of -innervated spindles may be preferentially to monitor mechanical disturbances arising from the activity of extrafusal muscle units with which they share motor innervation.     

Susceptibility ofBacteroides non-fragilis and fusobacteria to amoxicillin,amoxicillin/clavulanate,ticarcillin, ticarcillin/clavulanate,cefoxitin, imipenem and metronidazole

The susceptibility of 234Bacteroides non-fragilis strains and 56 fusobacteria from 12 European centers to amoxicillin, amoxicillin/clavulanate, ticarcillin, ticarcillin/clavulanate, cefoxitin, imipenem and metronidazole was tested and related to -lactamase production. Beta-lactamase production was detected in 42.3 % of theBacteroides strains and 26.8 % of the fusobacteria. The MIC90 of amoxicillin for -lactamase-negative strains was 0.5 µg/ml and the MIC90 of ticarcillin 2.0 µg/ml. In the case of -lactamase-positive strains the MIC90 of amoxicillin (32 µg/ml) and ticarcillin (16 µg/ml) dropped to 1.0 µg/ml upon addition of clavulanate; 65.8 % of these strains were susceptible to amoxicillin and 98.2 % to ticarcillin, but all were susceptible when clavulanate was added. All strains were susceptible to imipenem and metronidazole, and 99.3 % to cefoxitin.     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Phenotypic characteristics of lewis lung carcinoma cells

We studied adhesive properties and physiological activity in vivo of cells from Lewis lung carcinoma and its metastases. These cells differed in tumorogenic activity and metastatic potential in the syngeneic system. In vivo non-metastasizing cells are characterized by a lower content of surface lectins to tetrasaccharides SiaLe^x [Neu5Ac2-3Gal1-4(Fuc1-3) GlcNAc] and SiaLe^a [Neu5Ac2-3Gal1-3(Fuc1-4)GlcNAc] and trisaccharide HSO₃Le^x [HSO₃2-3Gal1-4(Fuc1-3)GlcNAc] compared to cells forming metastases in the syngeneic system. Metastatic cells with low tumorogenic activity weakly expressed lectins to disaccharide ligands 6-SiaLac [Neu5Ac2-6Gal1-4Glc], 6-HSO₃LacNAc, and A-di [GalNAc 1-3Gal] and trisaccharides H-type 1 [Fuc1-2Gal1-3GlcNAc and Le^x [Fuc1-3(Gal 1-4)GlcNAc] compared to cells that initiated tumor formation in the syngeneic system (similarly to transplanted tumors). We hypothesized that cell receptors to these carbohydrate determinates are involved in the development and growth of primary tumors, while lectins to SiaLe^x, SiaLe^a, and HSO₃Le^x play a role in the progress of tumor process and metastasizing.